Tumor supressor gene p33ING2

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of novel human tumor suppressors, antibodies to such tumor suppressors, methods of detecting such nucleic acids and proteins, methods of screening for modulators of tumor suppressors, and methods of diagnosing and treating tumors with such nucleic acids and proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S.Ser. No. 60/121,891, filed Feb. 26, 1999, the disclosure of which isherein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] This invention relates to isolated nucleic acid and amino acidsequences of novel human tumor suppressors, antibodies to such tumorsuppressors, methods of detecting such nucleic acids and proteins,methods of screening for modulators of tumor suppressors, and methods ofdiagnosing and treating tumors with such nucleic acids and proteins.

BACKGROUND OF THE INVENTION

[0004] Certain tumors, benign, premalignant, and malignant, are known tohave genetic components. Some of these tumors are caused by mutations orinactivation of “tumor suppressor” genes. In normal cells, the tumorsuppressor genes are involved in the regulation of cell growth andproliferation and in the control of cellular aging, anchorage dependenceand apoptosis. When the tumor suppressor genes are mutated orinactivated, cells are transformed and become immortalized ortumorigenic. These transformed cells can be reverted back to the normalphenotype (i.e., the cell growth rate is suppressed) by introducing thewildtype suppressor genes.

[0005] The first tumor suppressor gene identified was the nuclearphosphoprotein, retinoblastoma gene (Rb). Retinoblastoma is a malignanttumor of the sensory layer of the retina, and often occurs bilaterallyduring childhood. Retinoblastoma exhibits a familial tendency, but itcan be acquired. Mutations in the Rb gene and inactivation of itsproduct have been shown to be involved in other tumors, such as bladder,breast, small cell lung carcinomas, osteosarcomas, and soft tissuesarcomas. It was demonstrated that reconstitution of Rb-deficient tumorcells with the wildtype Rb leads to the suppression of growth rate ortumorigenicity (Huang et al., Science 242:1563-1566 (1988)). This resultprovides direct evidence that Rb protein is a tumor suppressor.

[0006] Another well-characterized tumor suppressor is the gene for thenuclear phosphoprotein, p53. More than half of all human cancers areassociated with mutations in the tumor suppressor gene p53 (see, e.g.,Hollstein et al., Science 253:49-53 (1991); Caron de Fronmentel &Soussi, Genes Chromosom. Cancer 4: 1-15; Harris & Hollstein, N. Engl. J.Med. 329:1318-1327 (1993); Greenblatt et al., Cancer Res. 54:4855-4878(1994)). Mutations in p53 often appear to be a critical step in thepathogenesis and progression of tumors. For example, missense mutationsof p53 occur in tumors of the colon, lung, breast, ovary, bladder, andseveral other organs. Alternatively, inactivation of the wildtype p53proteins in cells can cause tumors. For example, certain strains ofhuman papillomavirus (HPV) are known to interfere with the p53 proteinfunction, because the virus produces a protein, E6, which promotes thedegradation of the p53 protein.

[0007] Recently, another tumor suppressor gene, p33ING1, has beenidentified. p33ING1 directly cooperates with tumor suppressor gene p53in growth regulation (Garkavtsev et al., Nature Genetics 14:415-420(1996); Garkavtsev et al., Nature 391:295-298 (1998); U.S. Pat. No.5,986,078, all of which are herein incorporated by reference). Neitherof p53 or p33ING1 can alone cause growth inhibition when the other oneis suppressed (Garkavtsev et al. (1998), supra). According toimmunoprecipitation studies, p33ING1 proteins modulate the p53 activitythrough physical interaction. It has been also reported that someneuroblastoma cells have a mutation of the p33ING1 gene, and some breastcancer cell lines exhibit reduced expression of p33ING1 (Garkavtsev etal. (1996), supra).

[0008] Cancer remains a major public concern. Although epidemiologicaland cytogenetic studies demonstrated that a number of recessive geneticmutations are involved in various cancers, only a limited number oftumor suppressors have been identified. Therefore, there is a need toidentify and isolate other tumor suppressor genes. The identificationand isolation of new tumor suppressor genes would assist the diagnosis,prevention, and treatment of tumors and cancers.

SUMMARY OF THE INVENTION

[0009] The present invention thus provides for the first time nucleicacid and amino acid sequences of a new tumor suppressor gene calledp33ING2, as well as antibodies to p33ING2, methods of detecting suchnucleic acids and proteins, methods of screening for modulators ofp33ING2, and methods of diagnosing and treating tumors. P33ING2 nucleicacids and proteins are tumor suppressors that play a key role inregulation of cell proliferation and tumor suppression.

[0010] In one aspect, the present invention provides an isolated nucleicacid encoding a tumor suppressor polypeptide p33ING2, wherein thepolypeptide has greater than 70% amino acid sequence identity to apolypeptide comprising an amino acid sequence of SEQ ID NO:1. In oneembodiment, the nucleic acid encodes a polypeptide that selectivelybinds to polyclonal antibodies generated against a polypeptidecomprising an amino acid sequence of SEQ ID NO:1. In another embodiment,the nucleic acid encodes a polypeptide comprising an amino acid sequenceof SEQ ID NO:1. In yet another embodiment, the nucleic acid comprises anucleotide sequence of SEQ ID NO:2. In yet another embodiment, thenucleic acid is from human. In yet another embodiment, the nucleic acidis amplified by primers that selectively hybridize under stringenthybridization conditions to the same sequence as degenerate primer setsencoding amino acid sequences selected from the group consisting of: SEQID NO:3 (MLGQQQQ) and SEQ ID NO:4 (KKDRRSR). In yet another embodiment,the nucleic acid encodes a polypeptide having a molecular weight ofabout 28 kDa to about 38 kDa. In yet another embodiment, the nucleicacid encodes a tumor suppressor polypeptide p33ING2 that specificallyhybridizes under stringent conditions to a nucleic acid comprising anucleotide sequence of SEQ ID NO:2. In yet another embodiment, thenucleic acid selectively hybridizes under moderately stringenthybridization conditions to a nucleic acid comprising a nucleotidesequence of SEQ ID NO:2.

[0011] In another aspect, the present invention provides an isolatedtumor suppressor polypeptide p33ING2, wherein the polypeptide hasgreater than 70% amino acid sequence identity to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1. In one embodiment, thetumor suppressor polypeptide selectively binds to polyclonal antibodiesgenerated against a polypeptide comprising an amino acid sequence of SEQID NO:1. In another embodiment, the polypeptide is from human. In yetanother embodiment, the polypeptide is wildtype p33ING2.

[0012] In yet another aspect, the present invention provides an antibodythat selectively binds to a p33ING2 polypeptide comprising an amino acidsequence of SEQ ID NO:1, but does not bind to a p33ING1 polypeptidecomprising an amino acid sequence of SEQ ID NO:8. In one embodiment, theantibody is polyclonal. In another embodiment, the antibody selectivelybinds to a p33ING2 polypeptide comprising the amino acid sequence of SEQID NO:5, but does not bind to a p33ING1 polypeptide comprising an aminoacid sequence of SEQ ID NO:8.

[0013] In yet another aspect, the present invention provides anexpression vector comprising any one or more of the p33ING2 nucleic aciddescribed herein. The invention also provides a host cell transfectedwith a vector comprising any one or more of the p33ING2 nucleic aciddescribed herein.

[0014] In yet another aspect, the present invention provides a methodfor identifying a compound that modulates a tumor suppressor polypeptidep33ING2, the method comprising the steps of: (i) contacting the compoundwith a eukaryotic host cell or cell membrane in which has been expresseda tumor suppressor polypeptide p33ING2, wherein the polypeptide hasgreater than 70% amino acid sequence identity to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1; and (ii) determiningthe functional effect of the compound upon the cell or cell membraneexpressing the polypeptide. In one embodiment of the method, thepolypeptide selectively binds to polyclonal antibodies generated againsta polypeptide comprising an amino acid sequence of SEQ ID NO:1. Inanother embodiment of the method, functional effect is determined bymeasuring changes in cell growth. In yet another embodiment of themethod, the polypeptide is recombinant. In yet another embodiment of themethod, the polypeptide is from a human. In yet another embodiment ofthe method, the polypeptide comprises an amino acid sequence of SEQ IDNO:1. In yet another embodiment of the method, the cell is an HCT116human colon cancer cell line. In yet another embodiment of the method,the cell has the missense p33ING2 sequence of a polypeptide comprisingan amino acid sequence of SEQ ID NO:6.

[0015] In yet another aspect, the present invention provides a method ofinhibiting cellular proliferation, the method comprising transducing acell with an expression vector, the vector comprising a nucleic acidencoding a tumor suppressor polypeptide p33ING2, wherein the polypeptidehas greater than 70% amino acid sequence identity to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1. In one embodiment ofthe method, the polypeptide selectively binds to polyclonal antibodiesgenerated against a polypeptide comprising an amino acid sequence of SEQID NO:1. In another embodiment of the method, the nucleic acid encodes apolypeptide comprising an amino acid sequence of SEQ ID NO:1. In yetanother embodiment of the method, the nucleic acid comprises anucleotide sequence of SEQ ID NO:2. In yet another embodiment of themethod, the nucleic acid is from human. In yet another embodiment of themethod, the nucleic acid encodes a polypeptide having a molecular weightof about 28 kDa to about 38 kDa. In yet another embodiment of themethod, the cell has a missense or null endogenous p33ING2 phenotype. Inyet another embodiment of the method, the cell has a missense p33ING2sequence of a polypeptide comprising an amino acid sequence of SEQ IDNO:6.

[0016] In yet another aspect, the present invention provides a method ofdetecting the presence or absence of p33ING2 in mammalian tissue, themethod comprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING2-specific reagent thatselectively associates with p33ING2; and (iii) detecting the level ofp33ING2-specific reagent that selectively associates with the sample. Inone embodiment of the method, the p33ING2-specific reagent is selectedfrom the group consisting of a p33ING2-specific antibody, ap33ING2-specific primer, and a p33ING2-specific nucleic acid probe. Inanother embodiment of the method, the p33ING2-specific nucleic acidprobe binds to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:7, or to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:2, or to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:10. In yet another embodiment of the method, the biological samplecomprises intact chromosome 4q35. In yet another embodiment of themethod, the p33ING2-specific reagent detects nucleic acid, such as DNAor RNA. In yet another embodiment of the method, the nucleic acid is apolymorphic variant of p33ING2. In yet another embodiment of the method,the p33ING2-specific reagent is an antibody that selectively binds top33ING2. In some embodiments, the antibody is polyclonal. In yet anotherembodiments of the method, the antibody selectively binds to a p33ING2polypeptide comprising an amino acid sequence of SEQ ID NO:1, but not toa p33ING1 polypeptide comprising an amino acid sequence of SEQ ID NO:8.In yet another embodiment of the method, the antibody selectively bindsto a p33ING2 polypeptide comprising an amino acid sequence of SEQ IDNO:5, but does not bind to a p33ING1 polypeptide comprising an aminoacid sequence of SEQ ID NO:8.

[0017] In yet another aspect, the present invention provides a method ofdetermining a test amount of p33ING2 in mammalian tissue, the methodcomprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING2-specific reagent thatselectively associates with p33ING2; and (iii) comparing the test amountto a control. In one embodiment, the control is an amount of p33ING2 ina normal cell. In another embodiment, the p33ING2-specific reagent isselected from the group consisting of p33ING2-specific antibody, ap33ING2-specific primer; and p33ING2-specific nucleic acid probe.

[0018] In yet another aspect, the present invention provides a method ofdetecting the presence or absence of p33ING1 in mammalian tissue, themethod comprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING1-specific antibody thatselectively binds to p33ING1 but not to p33ING2; and

[0019] (iii) detecting the level of p33ING1-specific antibody thatselectively associates with the sample. In one embodiment, thep33ING1-specific antibody is polyclonal.

[0020] In yet another aspect, the present invention provides a method ofdetermining a test amount of p33ING1 in mammalian tissue, the methodcomprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING1-specific antibody thatselectively associates with p33ING1 but not to p33ING2; and (iii)comparing the test amount to a control. In one embodiment, the controlis an amount of p33ING1 in a normal cell. In another embodiment, thep33ING1-specific antibody is polyclonal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 illustrates binding specificities of polyclonal antibodiesfor p33ING2 and polyclonal antibodies for p33ING1 by ELISA.

[0022]FIG. 2 illustrates binding specificities of polyclonal antibodiesfor p33ING2 and polyclonal antibodies for p33ING1 by Western blotanalysis.

[0023]FIG. 3 illustrates that p33ING2 inhibits cell growth of HCT116cell line by colony formation assay.

[0024]FIG. 4 illustrates a Western blot that shows that p33ING2 proteinis induced by topoisomerase II inhibitor, etoposide.

[0025]FIG. 5 illustrates FACScan flow cytometric data that shows thatp33ING1 or p33ING2 can induce G₁ cell cycle arrest.

DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Introduction

[0027] The present invention provides for the first time nucleic acidsand polypeptides of a new tumor suppressor called p33ING2. The presentinvention also provides antibodies which selectively bind to a p33ING2protein, but not to a p33ING1 protein; and antibodies which selectivelybind to a p33ING1 protein, but not to a p33ING2 protein. These nucleicacids and the polypeptides they encode are tumor suppressors. Thesetumor suppressor nucleic acids and polypeptides are involved in theregulation of cell proliferation and in the control of cellular aging,anchorage dependence, and apoptosis.

[0028] The present invention also provides methods of screening formodulators (e.g. activators, inhibitors, stimulators, enhancers,agonists, and antagonists) of these novel p33ING2 proteins. Suchmodulators are useful for pharmacological and genetic modulation of cellgrowth and tumor suppression. The invention thus provides assays fortumor suppression and cell growth, where p33ING2 acts as a direct orindirect reporter molecule for measuring the effect of modulators oncell growth or tumor suppression. These assays can measure variousparameters that are affected by the p33ING2 activity, e.g., cell growthon soft agar, contact inhibition and density limitation of growth,growth factor or serum dependence, tumor specific markers levels,invasiveness into Matrigel, tumor growth in vivo, p33ING2 protein ormRNA levels, transcriptional activation or repression of a reportergene, and the like.

[0029] The present invention also provides methods of inhibiting cellproliferation of a cell by transducing the cell with an expressionvector containing p33ING2 nucleic acids. The transduced cell may have amissense or null endogenous p33ING2 phenotype or a mutation in anothertumor suppressor gene. For example, the cell may contain p33ING2 havinga sequence of SEQ ID NO:6 with a missense mutation. Expression ofwildtype p33ING2 restores cell growth regulation and prevents thedevelopment of tumor. For example, p33ING2 nucleic acids can be used totreat cancer or other cell proliferative diseases, such as hyperplasia,in patients.

[0030] Finally, the invention provides for methods of detecting p33ING2or p33ING1 nucleic acid and protein expression, allowing investigationof cell growth regulation and tumor suppression. Furthermore, p33ING2 orp33ING1 nucleic acid and protein expression can be used to diagnosecancer in patients who have a defect in one or more copies of p33ING2 orp33ING1 in their genome.

[0031] Functionally, p33ING2 represents a nuclear protein having amolecular weight of approximately 33 kDa. It is involved in theregulation of cell proliferation and in the control of cellular aging,anchorage and apoptosis.

[0032] Structurally, the nucleotide sequence of p33ING2 (see, e.g., SEQID NO:2, isolated from a human) encodes a polypeptide of approximately270 amino acids with a predicted molecular weight of approximately 33kDa and a predicted range of 28-38 kDa (see, e.g., SEQ ID NO:1). Relatedp33ING2 genes from other species share at least about 70% amino acididentity over an amino acid region of at least about 25 amino acids inlength, preferably 50 to 100 amino acids in length.

[0033] Specific regions of the p33ING2 nucleotide and amino acidsequences may be used to identify polymorphic variants, interspecieshomologs, and alleles of p33ING2. This identification can be made invitro, e.g., under stringent hybridization conditions or with PCR andsequencing, or by using the sequence information in a computer systemfor comparison with other nucleotide or amino acid sequences. Typically,identification of polymorphic variants and alleles of p33ING2 is made bycomparing an amino acid sequence of about 25 amino acids or more,preferably 50-100 amino acids. Amino acid identity of approximately atleast 70% or above, preferably 80%, most preferably 90-95% or abovetypically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of p33ING2. Sequence comparison can beperformed using any of the sequence comparison algorithms discussedbelow. Antibodies that bind specifically to p33ING2 or a conservedregion thereof can also be used to identify alleles, interspecieshomologs, and polymorphic variants.

[0034] Polymorphic variants, interspecies homologs, and alleles ofp33ING2 are confirmed by examining the effect of putative p33ING2expression on cell growth and tumor suppression using the methods andassays described herein. Typically, p33ING2 having the amino acidsequence of SEQ ID NO:1 is used as a positive control. For example,immunoassays using antibodies directed against the amino acid sequenceof SEQ ID NOS:1 or 5 can be used to demonstrate the identification of apolymorphic variant or allele of p33ING2. Alternatively, p33ING2 havingthe nucleic acid sequences of SEQ ID NO:1 is used as a positive control,e.g., in in situ hybridization with SEQ ID NO:1 to demonstrate theidentification of a polymorphic variant or allele of p33ING2. Thepolymorphic variants, alleles and interspecies homologs of p33ING2 areexpected to retain the ability to inhibit cell proliferation and tumorsuppression. These functional characteristics can be tested usingvarious assays, such as soft agar assay, contact inhibition and densitylimitation of growth assay, growth factor or serum dependence assay,tumor specific markers assay, invasiveness assay, apoptosis assay, G₀/G₁cell cycle arrest assay, tumor growth assay, etc.

[0035] The present invention also provides polymorphic variants ofp33ING2 depicted in SEQ ID NO:1: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 11;variant #2, in which a leucine residue is substituted for an isoleucineresidue at amino acid position 101; and variant #3, in which an alanineresidue is substituted for a glycine residue at amino acid position 251.

[0036] P33ING2 nucleotide and amino acid sequence information may alsobe used to construct models of tumor suppressor polypeptides in acomputer system. These models are subsequently used to identifycompounds that can activate or inhibit p33ING2. Such compounds thatmodulate the activity of p33ING2 can be used to investigate the role ofp33ING2 in inhibition of cell proliferation and tumor suppression or canbe used as therapeutics.

[0037] Isolation of p33ING2 provides a means for assaying for modulatorsof p33ING2. P33ING2 is useful for testing modulators using in vivo andin vitro expression that measure various parameters, e.g., cell growthon soft agar, contact inhibition and density limitation of growth,growth factor or serum dependence, tumor specific markers levels,invasiveness into Matrigel, apoptosis assay, G₀/G₁ cell cycle arrestassay, tumor growth in vivo, p33ING2 protein or mRNA levels,transcriptional activation or repression of a reporter gene, and thelike. Such modulators identified using p33ING2 can be used to study cellgrowth regulation and tumor suppression, and further to treat cancer.

[0038] Methods of detecting p33ING2 nucleic acids and expression ofp33ING2 are also useful for diagnosing various cancers or tumors byusing assays such as northern blotting, dot blotting, in situhybridization, RNase protection, and the like. Chromosome localizationof the genes encoding human p33ING2 can also be used to identifydiseases, mutations, and traits caused by and associated with p33ING2.Techniques, such as high density oligonucleotide arrays (GeneChip™), canbe also be used to screen for mutations, polymorphic variants, allelesand interspecies homologs of p33ING2.

[0039] II. Definitions

[0040] As used herein, the following terms have the meanings ascribed tothem unless specified otherwise.

[0041] The term “tumor suppressor” refers to a gene, or the protein itencodes, that in its wildtype form has the ability to suppress, prevent,or decrease cell transformation. Tumor suppressor genes are genes thatencode protein(s) that regulate cell growth and proliferation directlyor indirectly, e.g., p53, Rb, and the like. If a tumor suppressor geneis damaged (e.g., by radiation, a carcinogen or inherited, orspontaneous mutation), it may lose its wildtype ability to regulate cellgrowth and proliferation, and the cells may become transformed orpre-disposed to transformation.

[0042] “p33ING” refers to a family of tumor suppressor nucleic acids orpolypeptides having a molecular weight of approximately 33 kDa. Theyencode a nuclear protein which is involved in the regulation of cellgrowth and proliferation and in the control of cellular aging, anchorageand apoptosis. “p33ING2” and “p33ING1” are members of the “p33ING”family, which members are encoded by different genes (i.e., mapped todifferent regions on the chromosome). p33ING2 is mapped to humanchromosome 4q35.

[0043] The term p33ING2 therefore refers to polymorphic variants,alleles, interspecies homologs, and mutants that: (1) have about 70%amino acid sequence identity, preferably about 80-90% amino acidsequence identity to SEQ ID NO:1 over a window of about at least 50-100amino acids; (2) binds to polyclonal antibodies raised against animmunogen comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 and conservatively modified variants thereof,but does not bind to polyclonal antibodies raised against an immunogencomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:8 and conservatively modified variants thereof; (3)specifically hybridize (with a size of at least about 500, preferably atleast about 900 nucleotides) under stringent hybridization conditions toa sequence selected from the group consisting of SEQ ID NO:2, andconservatively modified variants thereof; or (4) are amplified byprimers that specifically hybridize under stringent conditions to thesame sequence as a degenerate primers sets encoding SEQ ID NOS:3 and 4.

[0044] The term p33ING1 refers to polymorphic variants, alleles,interspecies homologs, and mutants that: (1) have about 70% amino acidsequence identity, preferably about 80-90% amino acid sequence identityto SEQ ID NO:8 over a window of about at least 50-100 amino acids; (2)binds to polyclonal antibodies raised against an immunogen comprising anamino acid sequence selected from the group consisting of SEQ ID NO:8and conservatively modified variants thereof, but does not bind topolyclonal antibodies raised against an immunogen comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1 andconservatively modified variants thereof.

[0045] The phrases “polymorphic variant” and “allele” refer to forms ofp33ING2 that occur in a population (or among populations) and thatmaintain wildtype p33ING2 activity as measured using one of the assaysdescribed herein.

[0046] The term “mutant” of p33ING2 refers to those mutants which areexperimentally made or those which are found in tumor or cancer cells.Mutants of p33ING2 can be due to, e.g., truncation, elongation,substitution of amino acids, deletion, insertion, or lack of expression(e.g., due to promoter or splice site mutations, etc.). A mutant hasactivity that differs from the activity of wildtype p33ING2 by at leastabout 20% as measured using an assay described herein. For example, amutant of p33ING2 can have a null mutation which results in absence ofnormal gene product at the molecular level or an absence of function atthe phenotypic level. Another example is a missense mutation of p33ING2,where a substitution of amino acid(s) results in a change in theactivity of the protein.

[0047] The phrase “missense or null endogenous p33ING2 phenotype” of acell therefore refers to p33ING2 has a missense or null mutation so thatthe cell has a phenotype (e.g., soft agar growth, contact inhibition anddensity limitation of growth, etc.) which differs from a cell having awildtype p33ING2.

[0048] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0049] The term “transfect” or “transduce” refers to any way of gettinga nucleic acid across a cell membrane, including electroporation,biolistics, injection, plasmid transfection, lipofection, viraltransduction, lipid-nucleic acid complexes, naked DNA, etc

[0050] A “host cell” is a naturally occurring cell or a transformed cellthat contains an expression vector and supports the replication orexpression of the expression vector. Host cells may be cultured cells,explants, cells in vivo, and the like. Host cells may be prokaryoticcells such as E. coli, or eukaryotic cells such as yeast, insect,amphibian, or mammalian cells such as CHO, HeLa, HCT116, RKO cells, andthe like.

[0051] “Biological sample” include, but are not limited to, tissueisolated from humans, mice, and rats. In some embodiments, a sample ofbiological tissue or fluid contains nucleic acids or polypeptides ofp33ING2 and/or p33ING1. Biological samples may also include sections oftissues such as frozen sections taken from histological purposes. Abiological sample is typically obtained from a eukaryotic organism, suchas insects, protozoa, birds, fish, reptiles, and preferably a mammalsuch as rat, mouse, cow, dog, guinea pig, or rabbit, and most preferablya primate such as chimpanzees or humans.

[0052] “Tumor cell” refers to precancerous, cancerous, and normal cellsin a tumor.

[0053] “Cancer cells”, “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, and/or malignancy (see, Freshney, Culture of Animal Cells aManual of Basic Technique (3^(rd) ed. 1994)).

[0054] The term “cell cycle” refers to the cyclic biochemical andstructural evens occurring during growth of cells. The cell cycle isdivided into periods called: G₀, Gap₁, (G₁), DNA synthesis (S), GAP₂(G₂), and mitosis (M).

[0055] The phrase “functional effects” in the context of assays fortesting compounds that modulate p33ING2 mediated tumor suppressionincludes the determination of any parameter that is indirectly ordirectly under the influence of the p33ING2 protein. Functional effectsinclude, e.g., anchorage dependence, contact inhibition and densitylimitation of growth, growth factor or serum dependence, tumor specificmarkers levels, invasiveness, tumor growth, p33ING2 protein mRNA levels,apoptosis, G₀/G₁ cell cycle arrest, and the like, in vitro, in vivo, andex vivo.

[0056] By “determining the functional effect” is meant assays for acompound that increases or decreases a parameter that is directly orindirectly under the influence of p33ING2. Such functional effects canbe measured by any means known to those skilled in the art, e.g., softagar assay, contact inhibition and density limitation of growth assay,growth factor or serum dependence assay, tumor specific markers assay,invasiveness assay, apoptosis assay, G₀/G₁ cell cycle arrest assay,tumor growth assay, p33ING2 protein mRNA level assay, transcriptionalactivation or repression of a reporter gene assay, and the like, invitro, in vivo, and ex vivo.

[0057] “Inhibitors,” “activators,” and “modulators” of p33ING2 refer toinhibitory, activating, or modulatory molecules identified using invitro and in vivo assays for tumor suppression, e.g., ligands, agonists,antagonists, and their homologs and mimetics. Inhibitors are compoundsthat decrease, block, prevent, delay activation, inactivate,desensitize; or down regulate tumor suppression, e.g., antagonists.Activators are compounds that increase, activate, facilitate, enhanceactivation, sensitize or up regulate tumor suppression, e.g., agonists.Modulators are inhibitors and activators and include geneticallymodified versions of p33ING2, e.g., with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Such assays for modulators include,e.g., expressing p33ING2 in cells, applying putative modulatorcompounds, and then determining the functional effects on inhibition ofcell proliferation or tumor suppression. Compounds identified by theseassays are used to modulate tumor suppression effect of p33ING2.

[0058] Samples or assays comprising p33ING2 that are treated with apotential modulator are compared to control samples without theinhibitor, activator, or modulator. Control samples (untreated withinhibitors) are assigned a relative p33ING2 activity value of 100%.Inhibition of p33ING2 is achieved when the p33ING2 activity valuerelative to the control is about 90% or less, optionally about 80% orless, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less,or 25-0%. Activation of p33ING2 is achieved when the p33ING2 activityvalue relative to the control (untreated with activators) is 110% ormore, optionally 120%, 130%, 140%, 150% or more, 200-500% or more,1000-3000% or more.

[0059] The phrase “changes in cell growth” refers to any change in cellgrowth and proliferation characteristics in vitro or in vivo, such asformation of foci, anchorage independence, semi-solid or soft agargrowth, changes in contact inhibition and density limitation of growth,loss of growth factor or serum requirements, changes in cell morphology,gaining or losing immortalization, gaining or losing tumor specificmarkers, ability to form or suppress tumors when injected into suitableanimal hosts, and/or immortalization of the cell. See, e.g., Freshney,Culture of Animal Cells a Manual of Basic Technique, 3^(rd) ed.(Wiley-Liss, Inc. 1994), pp.231-241, herein incorporated by reference.The phrase “changes in cell growth” can also refer to changes inapoptosis or changes in cell cycle pattern.

[0060] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0061] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

[0062] A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

[0063] The term “operably linked” refers to a functional linkage betweena nucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

[0064] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0065] A “label” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available (e.g., the polypeptide of SEQ ID NO:1 can bemade detectable, e.g., by incorporating a radiolabel into the peptide,and used to detect antibodies specifically reactive with the peptide).

[0066] The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated p33ING2 nucleic acid is separated from openreading frames that flank the p33ING2 gene and encode proteins otherthan p33ING2. The term “purified” denotes that a nucleic acid or proteingives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

[0067] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0068] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

[0069] The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.

[0070] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group. Examples ofamino acid analogs include homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Amino acidmimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunction in a manner similar to a naturally occurring amino acid.

[0071] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0072] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences.Where the nucleic does not encode an amino acid sequence (e.g., aribosomal RNA), conservatively modified variants refer to essentiallyidentical sequences. Specifically, degenerate codon substitutions may beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

[0073] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0074] The following groups each contain amino acids that areconservative substitutions for one another:

[0075] 1) Alanine (A), Glycine (G);

[0076] 2) Serine (S), Threonine (T);

[0077] 3) Aspartic acid (D), Glutamic acid (E);

[0078] 4) Asparagine (N), Glutamine (Q);

[0079] 5) Cysteine (C), Methionine (M);

[0080] 6) Arginine (R), Lysine (K), Histidine (H);

[0081] 7) Isoleucine (I), Leucine (L), Valine (V); and

[0082] 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,Creighton, Proteins (1984)).

[0083] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity orhigher over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Preferably, the identity exists over aregion that is at least about 25 amino acids or nucleotides in length,or more preferably over a region that is 50-100 amino acids ornucleotides in length. In most preferred embodiments, the sequences aresubstantially identical over the entire length of, e.g., the codingregion.

[0084] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0085] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

[0086] A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N-4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff& Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0087] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0088] Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

[0089] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

[0090] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0091] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0092] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0093] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0094] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-temminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0095] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)—C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993)). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

[0096] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348, 552-554(1990); Marks et al., Biotechnology 10, 779-783 (1992)).

[0097] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0098] An “anti-p33ING2” antibody is an antibody or antibody fragmentthat specifically binds a polypeptide encoded by the p33ING2 gene, cDNA,or a subsequence thereof.

[0099] An “anti-p33ING1” antibody is an antibody or antibody fragmentthat specifically binds to a polypeptide encoded by the p33ING1 gene,cDNA, or a subsequence thereof.

[0100] The term “immunoassay” is an assay that uses an antibody tospecifically bind an antigen. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen.

[0101] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to p33ING2 atleast two times the background, more typically 10 to 1100 timesbackground, and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to a polyclonalantibody under such conditions may require an antibody that is selectedfor its specificity for a particular protein. For example, polyclonalantibodies raised to p33ING2 from a species such as rat, mouse, or humancan be selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with p33ING2 and not with other proteins,such as p33ING1, except for polymorphic variants and alleles of p33ING2.This selection may be achieved for polyclonal antibodies by subtractingout antibodies that cross react with p33ING1. For monoclonal antibodies,the specificity may be achieved by using a p33ING2 specific antigen tomake the hybridomas (e.g., SEQ ID NO:5). See, e.g., FIG. 2. Similarly,polyclonal antibodies raised to p33ING1 from a species such as rat,mouse, or human can be selected to obtain only those polyclonalantibodies that are specifically immunoreactive with p33ING1 and notwith other proteins, such as p33ING2, except for polymorphic variantsand alleles of p33ING1 using the methods described above. Foridentifying p33ING2 or p33ING1 variants and alleles from a particularspecies such as a human, the selection may be achieved by subtractingout antibodies that cross-react with p33ING2 or p33ING1 molecules,respectively, from other species. For species specific monoclonalantibodies, a species specific antigen can be used to make thehybridomas. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

[0102] The phrase “selectively associates with” refers to the ability ofa nucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

[0103] “p33ING2-specific reagent” refers to any reagent whichspecifically associates with p33ING2. For example, it can be ap33ING2-specific antibody, a p33ING2-specific primer, or ap33ING2-specific nucleic acid probe.

[0104] III. Isolation of the Gene Encoding p33ING2

[0105] A. General Recombinant DNA Methods

[0106] P33ING2 polypeptides and nucleic acids are used in the assaysdescribed below. For example, recombinant p33ING2 can be used to makecells that constitutively express p33ING2. Such polypeptides and nucleicacids can be made using routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2^(nd) ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

[0107] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

[0108] Oligonucleotides can be chernically synthesized according to thesolid phase phosphoramidite triester method first described by Beaucage& Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Purification of oligonucleotides is typically byeither native acrylamide gel electrophoresis or by anion-exchange HPLCas described in Pearson & Reanier, J. Chrom. 255:137-149 (1983). Thesequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981). Again, as noted above, companies such as Operon Technologies,Inc. provide an inexpensive commercial source for essentially anyoligonucleotide.

[0109] B. Cloning Methods for the Isolation of Nucleotide SequencesEncoding p33ING2

[0110] In general, the nucleic acid sequences encoding genes ofinterest, such as p33ING2 and related nucleic acid sequence homologs,are cloned from cDNA and genomic DNA libraries by hybridization with aprobe, or isolated using amplification techniques with oligonucleotideprimers. Preferably mammalian, more preferably human sequences are used.For example, p33ING2 sequences are typically isolated from mammaliannucleic acid (genomic or cDNA) libraries by hybridizing with a nucleicacid probe, the sequence of which can be derived from SEQ ID NO:1 Asuitable tissue from which human p33ING2 RNA and cDNA can be isolatedis, e.g., placenta, HepG2 or Saos-2 cell lines.

[0111] Amplification techniques using primers can also be used toamplify and isolate, e.g., a nucleic acid encoding p33ING2, from DNA orRNA (see, e.g., Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual(1995)). These primers can be used, e.g., to amplify either the fulllength sequence or a probe of one to several hundred nucleotides, whichis then used to screen a mammalian library for the full-length nucleicacid of choice. For example, degenerate primer sets, such as MLGQQQQ(SEQ ID NO:3) and KKDRRSR (SEQ ID NO:4) can be used to isolate p33ING2nucleic acids. Nucleic acids can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised, e.g., using the sequence of p33ING2.

[0112] Polymorphic variants and alleles that are substantially identicalto the gene of choice can be isolated using nucleic acid probes, andoligonucleotides under stringent hybridization conditions, by screeninglibraries. Alternatively, expression libraries can be used to clone,e.g., p33ING2 and p33ING2 polymorphic variants, interspecies homologs,and alleles, by detecting expressed homologs immunologically withantisera or purified antibodies made against p33ING2, which alsorecognize and selectively bind to the p33ING2 homolog.

[0113] To make a cDNA library, one should choose a source that is richin the mRNA of choice, e.g., for human p33ING2 mRNA, placenta, HepG2 orSaos-2 cell lines. The mRNA is then made into cDNA using reversetranscriptase, ligated into a recombinant vector, and transfected into arecombinant host for propagation, screening and cloning. Methods formaking and screening cDNA libraries are well known (see, e.g., Gubler &Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al.,supra).

[0114] For a genomic library, the DNA is extracted from the tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in non-lambdaexpression vectors. These vectors are packaged in vitro. Recombinantphage are analyzed by plaque hybridization as described in Benton &Davis, Science 196:180-182 (1977). Colony hybridization is carried outas generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA.,72:3961-3965 (1975).

[0115] An alternative method of isolating a nucleic acid and itshomologs combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of, e.g., p33ING2 directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify p33ING2 homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of p33ING2 encoding mRNA in physiologicalsamples, for nucleic acid sequencing, or for other purposes. Genesamplified by the PCR reaction can be purified from agarose gels andcloned into an appropriate vector.

[0116] As described above, gene expression of p33ING2 or p33ING1 canalso be analyzed by techniques known in the art, e.g., reversetranscription and PCR amplification of mRNA, isolation of total RNA orpoly A+ RNA, northern blotting, dot blotting, in situ hybridization,RNase protection, probing high density oligonucleotides, and the like.All of these techniques are standard in the art.

[0117] Synthetic oligonucleotides can be used to construct recombinantgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and non-sense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the p33ING2 nucleic acid. Thespecific subsequence is then ligated into an expression vector.

[0118] The nucleic acid encoding the protein of choice is typicallycloned into intermediate vectors before transformation into prokaryoticor eukaryotic cells for replication and/or expression. Theseintermediate vectors are typically prokaryote vectors, e.g., plasmids,or shuttle vectors. Optionally, cells can be transfected withrecombinant p33ING2 operably linked to a constitutive promoter, toprovide higher levels of p33ING2 expression in cultured cells.

[0119] C. Expression in Prokaryotes and Eukaryotes

[0120] To obtain high level expression of a cloned gene or nucleic acid,such as those cDNAs encoding p33ING2, one typically subclones p33ING2into an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the p33ING2 protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983)). Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available.

[0121] The promoter used to direct expression of a heterologous nucleicacid depends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function. Thepromoter typically cam also include elements that are responsive totransactivation, e.g., hypoxia responsive elements, Gal4 responsiveelements, lac repressor responsive elements, and the like. The promotercan be constitutive or inducible, heterologous or homologous.

[0122] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the nucleic acidin host cells. A typical expression cassette thus contains a promoteroperably linked, e.g., to the nucleic acid sequence encoding p33ING2,and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence may typically be linked to a cleavable signal peptide sequenceto promote secretion of the encoded protein by the transformed cell.Such signal peptides would include, among others, the signal peptidesfrom tissue plasminogen activator, insulin, and neuron growth factor,and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

[0123] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0124] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical (one expressionvector is described in Example I). Any of the conventional vectors usedfor expression in eukaryotic or prokaryotic cells may be used. Standardbacterial expression vectors include plasmids such as pBR322 basedplasmids, pSKF, pET23D, and fusion expression systems such as GST andLacZ. Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc.

[0125] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

[0126] Some expression systems have markers that provide geneamplification such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. Alternatively, high yield expressionsystems not involving gene amplification are also suitable, such asusing a baculovirus vector in insect cells, with a p33ING2 encodingsequence under the direction of the polyhedrin promoter or other strongbaculovirus promoters.

[0127] The elements that are typically included in expression vectorsalso include a replicon that functions in E. coli, a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Theparticular antibiotic resistance gene chosen is not critical, any of themany resistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

[0128] Standard transfection methods are used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

[0129] Any of the well known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, liposomes, microinjection, plasma vectors, viralvectors and any of the other well known methods for introducing clonedgenomic DNA, cDNA, synthetic DNA or other foreign genetic material intoa host cell (see, e.g., Sambrook et al., supra). It is only necessarythat the particular genetic engineering procedure used be capable ofsuccessfully introducing at least one gene into the host cell capable ofexpressing the protein of choice.

[0130] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe p33ING2 protein, which is recovered from the culture using standardtechniques identified below.

[0131] IV. Purification of p33ING2

[0132] If necessary, naturally occurring or recombinant proteins can bepurified for use in functional assays, e.g., to make antibodies todetect p33ING2. Naturally occurring p33ING2 is purified, e.g., frommammalian tissue such as placenta, HepG2 or Saos-2 cell lines or anyother source of a p33ING2 homolog. Recombinant p33ING2 is purified fromany suitable expression system, e.g., by expressing p33ING2 in E. coliand then purifying the recombinant protein via affinity purification,e.g., by using antibodies that recognize a specific epitope on theprotein or on part of the fusion protein, or by using glutathioneaffinity gel, which binds to GST. In some embodiments, the recombinantprotein is a fusion protein, e.g., with GST or Gal4 at the N-terminus.

[0133] The protein of choice may be purified to substantial purity bystandard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

[0134] A number of procedures can be employed when recombinant proteinis being purified. For example, proteins having established molecularadhesion properties can be reversibly fused to p33ING2. With theappropriate ligand, p33ING2 can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,p33ING2 could be purified using immunoaffinity columns.

[0135] A. Purification of p33ING2 from Recombinant Bacteria

[0136] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

[0137] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

[0138] If necessary, the inclusion bodies are solubilized, and the lysedcell suspension is typically centrifuged to remove unwanted insolublematter. Proteins that formed the inclusion bodies may be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. Other suitable buffers are known to those skilled in the art.The protein of choice is separated from other bacterial proteins bystandard separation techniques, e.g., with Ni—NTA agarose resin.

[0139] Alternatively, it is possible to purify the recombinant p33ING2protein from bacteria periplasm. After lysis of the bacteria, when theprotein is exported into the periplasm of the bacteria, the periplasmicfraction of the bacteria can be isolated by cold osmotic shock inaddition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

[0140] B. Standard Protein Separation Techniques for Purifying p33ING2

[0141] Solubility Fractionation

[0142] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0143] Size Differential Filtration

[0144] The molecular weight of the protein, e.g., p33ING2, can be usedto isolated the protein from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

[0145] Column Chromatography

[0146] The protein of choice can also be separated from other proteinson the basis of its size, net surface charge, hydrophobicity, andaffinity for ligands. In addition, antibodies raised against proteinscan be conjugated to column matrices and the proteins immunopurified.All of these methods are well known in the art. It will be apparent toone of skill that chromatographic techniques can be performed at anyscale and using equipment from many different manufacturers (e.g.,Pharmacia Biotech).

[0147] V. Immunological Detection of p33ING2 and p33ING1

[0148] In addition to the detection of p33ING2 genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect p33ING2, e.g., to identify alleles, mutants,polymorphic variants and interspecies homologs of p33ING2. Immunoassayscan be used to qualitatively or quantitatively analyze p33ING2, e.g., todetect p33ING2, to measure p33ING2 activity, or to identify modulatorsof p33ING2 activity. Similarly, immunoassay can be used to detect andanalyze p33ING1. A general overview of the applicable technology can befound in Harlow and Lane, Antibodies: A Laboratory Manual (1988).

[0149] A. Antibodies to p33ING2 and p33ING1

[0150] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with p33ING2 or p33ING1 are known to those of skillin the art (see, e.g., Coligan, Current Protocols in Immunology (1991);Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles andPractice (2^(nd) ed. 1986); and Kohler & Milstein, Nature 256:495-497(1975)). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). Inaddition, as noted above, many companies, such as BMA Biomedicals, Ltd.,HTI Bio-products, and the like, provide the commercial service of makingan antibody to essentially any peptide.

[0151] A number of p33ING2 or p33ING1 comprising immunogens may be usedto produce antibodies specifically reactive with p33ING2 or p33ING1,respectively. For example, recombinant p33ING2 or p33ING1, or antigenicfragments thereof, are isolated as described herein. Recombinant proteincan be expressed in eukaryotic or prokaryotic cells as described above,and purified as generally described above. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused an immunogen. Naturally occurring protein may also be used eitherin pure or impure form. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the protein.

[0152] Methods of production of polyclonal antibodies are known to thoseof skill in the art. To improve reproducibility, an inbred strain ofmice (e.g., BALB/C mice) can be immunized to make the antibody; however,standard animals (mice, rabbits, etc.) used to make antibodies areimmunized with the protein using a standard adjuvant, such as Freund'sadjuvant, and a standard immunization protocol (see Harlow & Lane,supra). The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and deterining the titer of reactivityto the protein of choice. When appropriately high titers of antibody tothe immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein can be done if desired (seeHarlow & Lane, supra).

[0153] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989).

[0154] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 10⁴ or greaterare selected and tested for their cross reactivity against non-p33ING2proteins or even other related proteins, e.g., from other organisms,using a competitive binding immunoassay. Specific polyclonal antiseraand monoclonal antibodies will usually bind with KD of at least about0.1 mM, more usually at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better.

[0155] Once p33ING2 or p33ING1 specific antibodies are available, theseproteins can be detected by a variety of immunoassay methods. For areview of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

[0156] B. Immunological Binding Assays

[0157] P33ING2 or p33ING1 can be detected and/or quantified using any ofa number of well recognized immunological binding assays (see, e.g.,U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For areview of the general immunoassays, see also Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunologicalbinding assays (or immunoassays) typically use an antibody thatspecifically binds to a protein or antigen of choice (in this casep33ING2, p33ING1, or antigenic fragments thereof). The antibody may beproduced by any of a number of means well known to those of skill in theart and as described above.

[0158] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeledp33ING2 or p33ING1 polypeptide or a labeled anti-p33ING2 or anti-p33ING1antibody. Alternatively, the labeling agent may be a third moiety, sucha secondary antibody, that specifically binds to the antibody/antigencomplex (a secondary antibody is typically specific to antibodies of thespecies from which the first antibody is derived). Other proteinscapable of specifically binding immunoglobulin constant regions, such asprotein A or protein G may also be used as the label agent. Theseproteins exhibit a strong non-immunogenic reactivity with immunoglobulinconstant regions from a variety of species (see, e.g., Kronval et al.,J. Immunol. 111: 1401-1406 (1973); Akerstrom et al., J. Immunol.135:2589-2542 (1985)). The labeling agent can be modified with adetectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

[0159] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0160] Non-Competitive Assay Formats

[0161] Immunoassays for detecting p33ING2 or p33ING1 in samples may beeither competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of antigen is directly measured. In onepreferred “sandwich” assay, for example, the anti-antigen antibodies canbe bound directly to a solid substrate on which they are immobilized.These immobilized antibodies then capture antigen present in the testsample. Antigen thus immobilized is then bound by a labeling agent, suchas a second antibody bearing a label. Alternatively, the second antibodymay lack a label, but it may, in turn, be bound by a labeled thirdantibody specific to antibodies of the species from which the secondantibody is derived. The second or third antibody is typically modifiedwith a detectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

[0162] Competitive Assay Formats

[0163] In competitive assays, the amount of p33ING2 or p33ING1 presentin the sample is measured indirectly by measuring the amount of a known,added (exogenous) antigen displaced (competed away) from an anti-antigenantibody by the unknown antigen present in a sample. In one competitiveassay, a known amount of antigen is added to a sample and the sample isthen contacted with an antibody that specifically binds to the antigen.The amount of exogenous antigen bound to the antibody is inverselyproportional to the concentration of antigen present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of antigen bound to the antibody may bedetermined either by measuring the amount of antigen present in anantigen/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of antigen may be detected byproviding a labeled antigen molecule.

[0164] A hapten inhibition assay is another preferred competitive assay.In this assay the known antigen is immobilized on a solid substrate. Aknown amount of anti-antigen antibody is added to the sample, and thesample is then contacted with the immobilized antigen. The amount ofanti-antigen antibody bound to the known immobilized antigen isinversely proportional to the amount of antigen present in the sample.Again, the amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

[0165] Cross-Reactivity Determinations

[0166] Immunoassays in the competitive binding format can also be usedfor crossreactivity determinations. For example, p33ING2 or p33ING1proteins can be immobilized to a solid support. Proteins are added tothe assay that compete for binding of the antisera to the immobilizedantigen. The ability of the added protein to compete for binding of theantisera to the immobilized protein is compared to the ability ofantigen to compete with itself. The percent crossreactivity for theabove proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with the added proteins areselected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the addedproteins.

[0167] Furthermore, immunoassays in the competitive binding format canbe used to determine cross-reactivity of polyclonal anti-p33ING2antibodies or p33ING1 antibodies for p33ING1 and p33ING2 proteins,respectively. As described above, p33ING2 protein can be immobilized toa solid support. p33ING1 protein is added to the assay, and the abilityof p33ING1 protein to compete for binding of the antisera to theimmobilized p33ING1 protein is compared to the ability of p33ING2 tocompete with itself. Those antisera with less than 10% crossreactivitywith p33ING1 protein are selected and pooled. Such immunoassays providesantibodies that selectively bind to a p33ING2 polypeptide but do notbind to a p33ING1 polypeptide. Similarly, immunoassays in thecompetitive binding format can be used to select antibodies thatselectively bind to a p33ING1 polypeptide, but do not bind to a p33ING2polypeptide. See, e.g., FIG. 2.

[0168] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein thought to be perhaps an allele, interspecies homologs, orpolymorphic variant of p33ING2 or p33ING1, to the immunogen protein. Inorder to make this comparison, the two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required to inhibit 50%of binding is less than 10 times the amount of the first protein that isrequired to inhibit 50% of binding, then the second protein is said tospecifically bind to the polyclonal antibodies generated to theimmunogen of choice.

[0169] Other Assay Formats

[0170] Western blot (immunoblot) analysis is used to detect and quantifythe presence of p33ING2 or p33ING1 in the sample. The techniquegenerally comprises separating sample proteins by gel electrophoresis onthe basis of molecular weight, transferring the separated proteins to asuitable solid support, (such as a nitrocellulose filter, a nylonfilter, or derivatized nylon filter), and incubating the sample with theantibodies that specifically bind p33ING2. The anti-antigen antibodiesspecifically bind to the antigen on the solid support. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the anti-antigen antibodies.

[0171] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

[0172] Reduction of Non-Specific Binding

[0173] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays. Particularlywhere the assay involves an antigen or antibody immobilized on a solidsubstrate, it is desirable to minimize the amount of non-specificbinding to the substrate. Means of reducing such non-specific bindingare well known to those of skill in the art. Typically, this techniqueinvolves coating the substrate with a proteinaceous composition. Inparticular, protein compositions such as bovine serum albumin (BSA),nonfat powdered milk, and gelatin are widely used with powdered milkbeing most preferred.

[0174] Labels

[0175] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0176] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0177] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize a specific protein, or secondary antibodies thatrecognize antibodies to the specific protein.

[0178] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0179] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0180] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0181] VI. Assays for Measuring Changes in p33ING2 Regulated Cell Growth

[0182] P33ING2 and its alleles, interspecies homologs, and polymorphicvariants participate in regulation of cell proliferation and tumorsuppression. Therefore, expression of p33ING2 and its alleles,interspecies homologs, and polymorphic variants in host cells wouldinhibit cell proliferation and suppress tumor formation. On the otherhand, expression of p33ING2 mutants in a cell could lead to abnormalcell proliferation and loss of tumor suppressor phenotypes. Finally,compounds that activate or inhibit p33ING2 would indirectly affectregulation of cellular proliferation and tumor suppression. Any of thesechanges in cell growth can be assessed by using a variety of in vitroand in vivo assays, e.g., ability to grow on soft agar, changes incontact inhibition and density limitation of growth, changes in growthfactor or serum dependence, changes in the level of tumor specificmarkers, changes in invasiveness into Matrigel, changes in apoptosis,changes in cell cycle pattern, changes in tumor growth in vivo, such asin transgenic mice, etc. Furthermore, these assays can be used to screenfor activators, inhibitors, and modulators of p33ING2. Such activators,inhibitors, and modulators of p33ING2 can then be used to modulatep33ING2 expression in tumor cells or abnormal proliferative cells.

[0183] A. Assays for Changes in Cell Growth by Expression of p33ING2Constructs

[0184] One or more of the following assays can be used to identifyp33ING2 constructs which are capable of regulating cell proliferationand tumor suppression. The phrase “p33ING2 constructs” can refer to anyof p33ING2 and its alleles, interspecies homologs, polymorphic variantsand mutants. Functional p33ING2 constructs identified by the followingassays can then be used in, e.g., gene therapy to inhibit abnormalcellular proliferation and transformation.

[0185] Soft Agar Growth or Colony Formation in Suspension

[0186] Normal cells require a solid substrate to attach and grow. Whenthe cells are transformed, they lose this phenotype and grow detachedfrom the substrate. For example, transformed cells can grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft agar. The transformed cells, when transfected with tumorsuppressor genes, regenerate normal phenotype and require a solidsubstrate to attach and grow.

[0187] Soft agar growth or colony formation in suspension assays can beused to identify p33ING2 constructs, which when expressed in host cells,inhibit abnormal cellular proliferation and transformation. Typically,transformed host cells (e.g., cells that grow on soft agar) are used inthis assay. For example, RKO or HCT116 cell lines can be used.Expression of a tumor suppressor gene in these transformed host cellswould reduce or eliminate the host cells' ability to grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft. This is because the host cells would regenerate anchoragedependence of normal cells, and therefore require a solid substrate togrow. Therefore, this assay can be used to identify p33ING2 constructsthat encode a functional tumor suppressor. Once identified, such p33ING2constructs can be used in a number of diagnostic or therapeutic methods,e.g., in gene therapy to inhibit abnormal cellular proliferation andtransformation.

[0188] Techniques for soft agar growth or colony formation in suspensionassays are described in Freshney, Culture of Animal Cells a Manual ofBasic Technique, 3^(rd) ed., Wiley-Liss, New York (1994), hereinincorporated by reference. See also, the methods section of Garkavtsevet al. (1996), supra, herein incorporated by reference.

[0189] Contact Inhibition and Density Limitation of Growth

[0190] Normal cells typically grow in a flat and organized pattern in apetri dish until they touch other cells. When the cells touch oneanother, they are contact inhibited and stop growing. When cells aretransformed, however, the cells are not contact inhibited and continueto grow to high densities in disorganized foci. Thus, the transformedcells grow to a higher saturation density than normal cells. This can bedetected morphologically by the formation of a disoriented monolayer ofcells or rounded cells in foci within the regular pattern of normalsurrounding cells. Alternatively, labeling index with [³H]-thymidine atsaturation density can be used to measure density limitation of growth.See Freshney (1994), supra. The transformed cells, when transfected withtumor suppressor genes, regenerate a normal phenotype and become contactinhibited and would grow to a lower density.

[0191] Contact inhibition and density limitation of growth assays can beused to identify p33ING2 constructs which are capable of inhibitingabnormal proliferation and transformation in host cells. Typically,transformed host cells (e.g., cells that are not contact inhibited) areused in this assay. For example, RKO or HCT116 cell lines can be used.Expression of a tumor suppressor gene in these transformed host cellswould result in cells which are contact inhibited and grow to a lowersaturation density than the transformed cells. Therefore, this assay canbe used to identify p33ING2 constructs which function as a tumorsuppressor. Once identified, such p33ING2 constructs can be used, e.g.,in gene therapy to inhibit abnormal cellular proliferation andtransformation.

[0192] In this assay, labeling index with [³H]-thymidine at saturationdensity is a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a p33ING2 construct and aregrown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with [³H]-thymidine isdetermined autoradiogrpahically. See, Freshney (1994), supra. The hostcells expressing a functional p33ING2 construct would give arise to alower labeling index compared to control (e.g., transformed host cellstransfected with a vector lacking an insert).

[0193] Growth Factor or Serum Dependence

[0194] Growth factor or serum dependence can be used as an assay toidentify functional p33ING2 constructs. Transformed cells have a lowerserum dependence than their normal counterparts (see, e.g., Temin, J.Natl. Cancer Insti. 37:167-175 (1966); Eagle et al., J. Exp. Med.131:836-879 (1970)); Freshney, supra. This is in part due to release ofvarious growth factors by the transformed cells. When a tumor suppressorgene is transfected and expressed in these transformed cells, the cellswould reacquire serum dependence and would release growth factors at alower level. Therefore, this assay can be used to identify p33ING2constructs which encode functional tumor suppressor. Growth factor orserum dependence of transformed host cells which are transfected with ap33ING2 construct can be compared with that of control (e.g.,transformed host cells which are transfected with a vector withoutinsert). Host cells expressing a functional p33ING2 would exhibit anincrease in growth factor and serum dependence compared to control.

[0195] Tumor Specific Markers Levels

[0196] Tumor cells release an increased amount of certain factors(hereinafter ‘tumor specific markers’) than their normal counterparts.For example, plasminogen activator (PA) is released from human glioma ata higher level than from normal brain cells (see, e.g., Gullino,Angiogenesis, tumor vascularization, and potential interference withtumor growth. In Mihich (ed.): “Biological Responses in Cancer.” NewYork, Academic Press, pp. 178-184 (1985)). Similarly, Tumor angiogenesisfactor (TAF) is released at a higher level in tumor cells than theirnormal counterparts. See, e.g., Folkman, Angiogenesis and cancer, SemCancer Biol. (1992)).

[0197] Tumor specific markers can be assayed for to identify p33ING2constructs, which when expressed, decrease the level of release of thesemarkers from host cells. Typically, transformed or tumorigenic hostcells are used. Expression of a tumor suppressor gene in these hostcells would reduce or eliminate the release of tumor specific markersfrom these cells. Therefore, this assay can be used to identify p33ING2constructs that encode a functional tumor suppressor.

[0198] Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980);Gulino, Angiogenesis, tumor vascularization, and potential interferencewith tumor growth. In Mihich, E. (ed): “Biological Responses in Cancer.”New York, Plenum (1985); Freshney Anticancer Res. 5:111-130 (1985).

[0199] Invasiveness into Matrigel

[0200] The degree of invasiveness into Matrigel or some otherextracellular matrix constituent can be used as an assay to identifyp33ING2 constructs which are capable of inhibiting abnormal cellproliferation and tumor growth. Tumor cells exhibit a good correlationbetween malignancy and invasiveness of cells into Matrigel or some otherextracellular matrix constituent. In this assay, tumorigenic cells aretypically used as host cells. Expression of a tumor suppressor gene inthese host cells would decrease invasiveness of the host cells.Therefore, functional p33ING2 constructs can be identified by measuringchanges in the level of invasiveness between the host cells before andafter the introduction of p33ING2 constructs. If a p33ING2 constructfunctions as a tumor suppressor, its expression in tumorigenic hostcells would decrease invasiveness.

[0201] Techniques described in Freshney (1994), supra, can be used.Briefly, the level of invasion of host cells can be measured by usingfilters coated with Matrigel or some other extracellular matrixconstituent. Penetration into the gel, or through to the distal side ofthe filter, is rated as invasiveness, and rated histologically by numberof cells and distance moved, or by prelabeling the cells with ¹²⁵I andcounting the radioactivity on the distal side of the filter or bottom ofthe dish. See, e.g., Freshney (1984), supra.

[0202] Apoptosis Analysis

[0203] Apoptosis analysis can be used as an assay to identify functionalp33ING2 constructs. p33ING2 expression or overexpression causesapoptosis (see Example IX below). In this assay, cell lines, such as RKOor HCT116, can be used to screen p33ING2 constructs which encode afunctional tumor suppressor. Cells are transfected with a putativep33ING2 construct. The cells can be co-transfected with a constructcomprising a marker gene, such as a gene that encodes green fluorescentprotein. Alternatively, a single construct comprising a putative p33ING2gene and a marker gene can be transfected into cells. Overexpression ofa p33ING2 gene that encodes a functional tumor suppressor would causeapoptosis. Not wishing to be bound by a theory, exogenous expression ofa tumor suppressor can decrease cell proliferation by causing a cellcycle arrest and by increasing cell death. The apoptotic change can bedetermined using methods known in the art, such as DAPI staining andTUNEL assay using fluorescent microscope. For TUNEL assay, commerciallyavailable kit can be used (e.g., Fluorescein FragEL DNA FragmentationDetection Kit (Oncogene Research Products, Cat.#QIA39)+Tetramethyl-rhodamine-5-dUTP (Roche, Cat. # 1534 378)). Cellsexpressing a functional p33ING2 would exhibit an increased apoptosiscompared to control (e.g., a cell transfected with a vector without ap33ING2 gene insert).

[0204] G₀/G₁ Cell Cycle Arrest Analysis

[0205] G₀/G₁ cell cycle arrest can be used as an assay to identifyfunctional p33ING2 constructs. p33ING2 expression or overexpressioncauses G1 cell cycle arrest (see Example IX below). In this assay, celllines, such as RKO or HCT116, can be used to screen p33ING2 constructswhich encode a functional tumor suppressor. Cells are transfected with aputative p33ING2 construct. The cells can be co-transfected with aconstruct comprising a marker gene, such as a gene that encodes greenfluorescent protein. Alternatively, a single construct comprising aputative p33ING2 gene and a marker gene can be transfected into cells.Expression or overexpression of a p33ING2 gene that encodes a functionaltumor suppressor would cause G₀/G₁ cell cycle arrest (see, e.g., ExampleVII). Methods known in the art can be used to measure the degree of G₁cell cycle arrest. For example, the propidium iodide signal can be usedas a measure for DNA content to determine cell cycle profiles on a flowcytometer. The percent of the cells in each cell cycle can becalculated. Cells expressing a functional p33ING2 would exhibit a highernumber of cells that are arrested in G₀/G₁ phase compared to control(e.g., transfected with a vector without a p33ING2 gene insert).

[0206] Tumor Growth In Vivo

[0207] Effects of p33ING2 on cell growth can be tested in transgenic orimmune-suppressed mice. Knock-out transgenic mice can be made, in whichthe endogenous p33ING2 gene is disrupted. Such knock-out mice can beused to study effects of p33ING2, e.g., as a cancer model, as a means ofassaying in vivo for compounds that modulate p33ING2, and to test theeffects of restoring a wildtype p33ING2 to a knock-out mice.

[0208] Knock-out transgenic mice can be made by insertion of a markergene or other heterologous gene into the endogenous p33ING2 gene site inthe mouse genome via homologous recombination. Such mice can also bemade by substituting the endogenous p33ING2 with a mutated version ofp33ING2, or by mutating the endogenous p33ING2, e.g., by exposure tocarcinogens.

[0209] A DNA construct is introduced into the nuclei of embryonic stemcells. Cells containing the newly engineered genetic lesion are injectedinto a host mouse embryo, which is re-implanted into a recipient female.Some of these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

[0210] These knock-out mice can be used as hosts to test the effects ofvarious p33ING2 constructs on cell growth. These transgenic mice withthe endogenous p33ING2 gene knocked out would develop abnormal cellproliferation and tumor growth. They can be used as hosts to test theeffects of various p33ING2 constructs on cell growth. For example,introduction of wildtype p33ING2 into these knock-out mice would inhibitabnormal cellular proliferation and suppress tumor growth.

[0211] Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, genetically athymic “nude” mouse (see,e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCIDmouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradleyet al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52(1980)) can be used as a host. Transplantable tumor cells (typicallyabout 10⁶ cells) injected into isogenic hosts will produce invasivetumors in a high proportions of cases, while normal cells of similarorigin will not. In hosts which developed invasive tumors, cellsexpressing a p33ING2 construct are injected subcutaneously. After asuitable length of time, preferably 4-8 weeks, tumor growth is measured(e.g., by volume or by its two largest dimensions) and compared to thecontrol. Tumors that have statistically significant reduction (using,e.g., Student's T test) are said to have inhibited growth. Usingreduction of tumor size as an assay, functional p33ING2 constructs whichare capable of inhibiting abnormal cell proliferation can be identified.This model can also be used to identify mutant versions of p33ING2.

[0212] B. Assays for Compounds that Modulate p33ING2

[0213] P33ING2 and its alleles, interspecies homologs, and polymorphicvariants participate in regulation of cell proliferation and tumorsuppression. Mutations in these genes, including null or missensemutations, can cause abnormal cell proliferation and tumor growth. Theactivity of p33ING2 polypeptides (wildtype or mutants) can be assessedusing a variety of in vitro and in vivo assays measuring variousparameters, e.g., cell growth on soft agar, contact inhibition anddensity limitation of growth, growth factor or serum dependence, tumorspecific markers levels, invasiveness into Matrigel, tumor growth invivo, transgenic mice, p33ING2 protein or mRNA levels, transcriptionalactivation or repression of a reporter gene, apoptosis analysis, G₀/G₁cell cycle arrest, and the like. Such assays can also be used to screenfor activators, inhibitors, and modulators of wildtype and mutantp33ING2. Such activators, inhibitors, and modulators are useful ininhibiting tumor growth and modulating cell proliferation. Compoundsidentified using the assays of the invention are useful as therapeuticsfor treatment of cancer and other diseases involving cellularhyperproliferation.

[0214] Biologically active or inactivated p33ING2 polypeptides, eitherrecombinants or naturally occurring, are used to screen activators,inhibitors, or modulators of tumor suppression and cell proliferation.The p33ING2 polypeptides can be recombinantly expressed in a cell,naturally expressed in a cell, recombinantly or naturally expressed incells transplanted into an animal, or recombinantly or naturallyexpressed in a transgenic animal. Modulation is tested using one of thein vitro or in vivo assays described in herein in part A.

[0215] Cells that have wildtype p33ING2, p33ING2 null mutations, p33ING2missense mutations, or inactivation of p33ING2 are used in the assays ofthe invention, both in vitro and in vivo. Preferably, human cells areused. Cell lines can also be created or isolated from tumors that havemutant p33ING2. Optionally, the cells can be transfected with anexogenous p33ING2 gene operably linked to a constitutive promoter, toprovide higher levels of p33ING2 expression. Alternatively, endogenousp33ING2 levels can be examined. The cells can be treated to inducep33ING2 expression. The cells can be immobilized, be in solution, beinjected into an animal, or be naturally occurring in a transgenic ornon-transgenic animal.

[0216] Samples or assays that are treated with a test compound whichpotentially activates, inhibits, or modulates p33ING2 are compared tocontrol samples that are not treated with the test compound, to examinethe extent of modulation. Generally, the compounds to be tested arepresent in the range from 0.11 nM to 10 mM. Control samples (untreatedwith activators, inhibitors, or modulators) are assigned relativep33ING2 activity value of 100%. Inhibition of p33ING2 is achieved whenthe p33ING2 activity value relative to the control is about 90% (e.g.,10% less than the control), optionally 80% or less, 70% or less, 60% orless, 50% or less, 40% or less, or 25-0%. Activation of p33ING2 isachieved when the p33ING2 activity value relative to the control is 110%or more (e.g., at least 10% more than the control), optionally 120%,130%, 140%, 150% or more, 200-500% or more, 1000-3000% or more.

[0217] The effects of the test compounds upon the function of thep33ING2 polypeptides can be measured by examining any of the parametersdescribed above. For example, parameters such as ability to grow on softagar, contact inhibition and density limitation of growth, growth factoror serum dependence, tumor specific markers levels, invasiveness intoMatrigel, apoptosis, G₀/G₁ cell cycle arrest, tumor growth in vivo,transgenic mice and the like, can be measured. Furthermore, the effectsof the test compounds on p33ING2 protein or mRNA levels, transcriptionalactivation or repression of a reporter gene can be measured. In eachassay, cells expressing p33ING2 are contacted with a test compound andincubated for a suitable amount of time, e.g., from 0.5 to 48 hours.Then, parameters such as those described above are compared to thoseproduced by control cells untreated with the test compound.

[0218] In one embodiment, the effect of test compounds upon the functionof p33ING2 can be determined by comparing the level of p33ING2 proteinor mRNA in treated samples and control samples. The level of p33ING2protein is measured using immunoassays such as western blotting, ELISAand the like with a p33ING2 specific antibody. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,northern hybridization, RNase protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

[0219] Alternatively, a reporter gene system can be devised using thep33ING2 promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or β-gal. After treatment with apotential p33ING2 modulator, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

[0220] In another embodiment, the effects of test compounds on p33ING2activity is performed in vivo. In this assay, cultured cells that areexpressing a wildtype or mutant p33ING2 (e.g., a null or missensemutation) are injected subcutaneously into an immune compromised mousesuch as an athymic mouse, an irradiated mouse, or a SCID mouse. P33ING2modulators are administered to the mouse, e.g., a chemical ligandlibrary. After a suitable length of time, preferably 4-8 weeks, tumorgrowth is measured, e.g., by volume or by its two largest dimensions,and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth. Alternatively, the extent of tumor neovascularization can alsobe measured. Immunoassays using endothelial cell specific antibodies areused to stain for vascularization of the tumor and the number of vesselsin the tumor. Tumors that have a statistically significant reduction inthe number of vessels (using, e.g., Student's T test) are said to haveinhibited neovascularization.

[0221] Alternatively, transgenic mice with the endogenous p33ING2 geneknocked out can be used in an assay to screen for compounds whichmodulate the p33ING2 activity. As described in part A, knock-outtransgenic mice can be made, in which the endogenous p33ING2 gene isdisrupted, e.g., by replacing it with a marker gene. A transgenic mousethat is heterozygous or homozygous for integrated transgenes that havefunctionally disrupted the endogenous p33ING2 gene can be used as asensitive in vivo screening assay for p33ING2 ligands and modulators ofp33ING2 activity.

[0222] C. Modulators

[0223] The compounds tested as modulators of p33ING2 can be any smallchemical compound, or a biological entity, such as a protein, sugar,nucleic acid or lipid. Alternatively, modulators can be geneticallyaltered versions of p33ING2. For example, an antisense construct ofp33ING2 can be used as a modulator.

[0224] Typically, test compounds will be small chemical molecules andpeptides. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although most oftencompounds can be dissolved in aqueous or organic (especially DMSO-based)solutions are used. The assays are designed to screen large chemicallibraries by automating the assay steps and providing compounds from anyconvenient source to assays, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). It willbe appreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

[0225] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0226] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0227] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

[0228] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0229] D. Solid State and Soluble High Throughput Assays

[0230] In one embodiment the invention provide soluble assays usingmolecules such as a domain such as ligand binding domain, an activesite, etc.; a domain that is covalently linked to a heterologous proteinto create a chimeric molecule; p33ING2; a cell or tissue expressingp33ING2, either naturally occurring or recombinant. In anotherembodiment, the invention provides solid phase based in vitro assays ina high throughput format, where the domain, chimeric molecule, p33ING2,or cell or tissue expressing p33ING2 is attached to a solid phasesubstrate.

[0231] In the high throughput assays of the invention, it is possible toscreen up to several thousand different modulators or ligands in asingle day. In particular, each well of a microtiter plate can be usedto run a separate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-1500 differentcompounds. It is possible to assay several different plates per day;assay screens for up to about 6,000-20,000 different compounds ispossible using the integrated systems of the invention.

[0232] The molecule of interest can be bound to the solid statecomponent, directly or indirectly, via covalent or non covalent linkage,e.g., via a tag. The tag can be any of a variety of components. Ingeneral, a molecule which binds the tag (a tag binder) is fixed to asolid support, and the tagged molecule of interest is attached to thesolid support by interaction of the tag and the tag binder.

[0233] A number of tags and tag binders can be used, based upon knownmolecular interactions well described in the literature. For example,where a tag has a natural binder, for example, biotin, protein A, orprotein G, it can be used in conjunction with appropriate tag binders(avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin,etc.) Antibodies to molecules with natural binders such as biotin arealso widely available and appropriate tag binders; see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.

[0234] Similarly, any haptenic or antigenic compound can be used incombination with an appropriate antibody to form a tag/tag binder pair.Thousands of specific antibodies are commercially available and manyadditional antibodies are described in the literature. For example, inone common configuration, the tag is a first antibody and the tag binderis a second antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

[0235] Synthetic polymers, such as polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, and polyacetates can also form anappropriate tag or tag binder. Many other tag/tag binder pairs are alsouseful in assay systems described herein, as would be apparent to one ofskill upon review of this disclosure.

[0236] Common linkers such as peptides, polyethers, and the like canalso serve as tags, and include polypeptide sequences, such as poly glysequences of between about 5 and 200 amino acids. Such flexible linkersare known to persons of skill in the art. For example, poly(ethyleneglycol) linkers are available from Shearwater Polymers, Inc. Huntsville,Ala. These linkers optionally have amide linkages, sulfhydryl linkages,or heterofunctional linkages.

[0237] Tag binders are fixed to solid substrates using any of a varietyof methods currently available. Solid substrates are commonlyderivatized or functionalized by exposing all or a portion of thesubstrate to a chemical reagent which fixes a chemical group to thesurface which is reactive with a portion of the tag binder. For example,groups which are suitable for attachment to a longer chain portion wouldinclude amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanesand hydroxyalkylsilanes can be used to functionalize a variety ofsurfaces, such as glass surfaces. The construction of such solid phasebiopolymer arrays is well described in the literature. See, e.g.,Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solidphase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth.102:259-274 (1987) (describing synthesis of solid phase components onpins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describingsynthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759(1996) (all describing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

[0238] E. Computer-Based Assays

[0239] Yet another assay for compounds that modulate p33ING2 activityinvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of p33ING2 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

[0240] The three-dimensional structural model of the protein isgenerated by entering p33ING2 amino acid sequences of at least 10 aminoacid residues or corresponding nucleic acid sequences encoding a p33ING2polypeptide into the computer system. The amino acid sequence of thepolypeptide or the nucleic acid encoding the polypeptide is selectedfrom the group consisting of SEQ ID NO:1 or SEQ ID NO:2, andconservatively modified versions thereof. The amino acid sequencerepresents the primary sequence or subsequence of the protein, whichencodes the structural information of the protein. At least 10 residuesof the amino acid sequence (or a nucleotide sequence encoding 10 aminoacids) are entered into the computer system from computer keyboards,computer readable substrates that include, but are not limited to,electronic storage media (e.g., magnetic diskettes, tapes, cartridges,and chips), optical media (e.g., CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe protein is then generated by the interaction of the amino acidsequence and the computer system, using software known to those of skillin the art. The three-dimensional structural model of the protein can besaved to a computer readable form and be used for further analysis(e.g., identifying potential ligand binding regions of the protein andscreening for mutations, alleles and interspecies homologs of the gene).

[0241] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary andquaternary structure of the protein of interest. The software looks atcertain parameters encoded by the primary sequence to generate thestructural model. These parameters are referred to as “energy terms,”and primarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

[0242] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0243] Once the structure has been generated, potential ligand bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of the p33ING2 protein to identify ligands that bind top33ING2. Binding affinity between the protein and ligands is determinedusing energy terms to determine which ligands have an enhancedprobability of binding to the protein. The results, such asthree-dimensional structures for potential ligands and binding affinityof ligands, can also be saved to a computer readable form and can beused for further analysis (e.g., generating a three dimensional model ofmutated proteins having an altered binding affinity for a ligand).

[0244] Computer systems are also used to screen for mutations,polymorphic variants, alleles and interspecies homologs of p33ING2genes. Such mutations can be associated with disease states or genetictraits. As described above, high density oligonucleotide arrays(GeneChip™) and related technology can also be used to screen formutations, polymorphic variants, alleles and interspecies homologs. Oncethe variants are identified, diagnostic assays can be used to identifypatients having such mutated genes. Identification of the mutatedp33ING2 genes involves receiving input of a first nucleic acid or aminoacid sequence encoding selected from the group consisting of SEQ IDNO:2, or SEQ ID NO:1, and conservatively modified versions thereof. Thesequence is entered into the computer system as described above and thensaved to a computer readable form. The first nucleic acid or amino acidsequence is then compared to a second nucleic acid or amino acidsequence that has substantial identity to the first sequence. The secondsequence is entered into the computer system in the manner describedabove. Once the first and second sequences are compared, nucleotide oramino acid differences between the sequences are identified. Suchsequences can represent allelic differences in p33ING2 genes, andmutations associated with disease states and genetic traits.

[0245] VII. Gene Therapy

[0246] The present invention provides the nucleic acids of p33ING2 forthe transfection of cells in vitro and in vivo. These nucleic acids canbe inserted into any of a number of well known vectors for thetransfection of target cells and organisms as described below. Thenucleic acids are transfected into cells, ex vivo or in vivo, throughthe interaction of the vector and the target cell. The nucleic acidsencoding p33ING2, under the control of a promoter, then expresses ap33ING2 of the present invention, thereby mitigating the effects ofabsent, partial inactivation, or abnormal expression of the p33ING2gene.

[0247] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and viral infection in a numberof contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies (for a review of gene therapy procedures, see Anderson,Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932(1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology (Doerfler & Böhmeds., 1995); and Yu et al., Gene Therapy 1: 13-26 (1994)).

[0248] Delivery of the gene or genetic material into the cell is thefirst critical step in gene therapy treatment of disease. A large numberof delivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon,TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460 (1992); VanBrunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

[0249] Methods of non-viral delivery of nucleic acids includelipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in, e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787;and U.S. Pat. No. 4,897,355) and lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids that are suitable for efficient receptor-recognition lipofectionof polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

[0250] The preparation of lipid:nucleic acid complexes, includingtargeted liposomes such as immunolipid complexes, is well known to oneof skill in the art (see, e.g., Crystal, Science 270:404-410 (1995);Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad etal., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183,4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,4,837,028, and 4,946,787).

[0251] The use of RNA or DNA viral based systems for the delivery ofnucleic acids take advantage of highly evolved processes for targeting avirus to specific cells in the body and trafficking the viral payload tothe nucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

[0252] The tropism of a retrovirus can be altered by incorporatingforeign envelope proteins, expanding the potential target population oftarget cells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

[0253] In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

[0254] In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

[0255] pLASN and MFG-S are examples are retroviral vectors that havebeen used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995);Kohn et al., Nat. Med. 1: 1017-102 (1995); Malech et al., Proc. Natl.Acad. Sci. U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the firsttherapeutic vector used in a gene therapy trial. (Blaese et al., Science270:475-480 (1995)). Transduction efficiencies of 50% or greater havebeen observed for MFG-S packaged vectors. (Ellem et al., ImmunolImmunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2(1997).

[0256] Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Keams et al., Gene Ther.9:748-55 (1996)).

[0257] Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 24:15-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

[0258] Packaging cells are used to form virus particles that are capableof infecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

[0259] In many gene therapy applications, it is desirable that the genetherapy vector be delivered with a high degree of specificity to aparticular tissue type. A viral vector is typically modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the viruses outer surface. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. U.S.A. 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., Fab or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

[0260] Gene therapy vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

[0261] Ex vivo cell transfection for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transfected cells into the hostorganism) is well known to those of skill in the art. In a preferredembodiment, cells are isolated from the subject organism, transfectedwith a nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

[0262] In one embodiment, stem cells are used in ex vivo procedures forcell transfection and gene therapy. The advantage to using stem cells isthat they can be differentiated into other cell types in vitro, or canbe introduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-αare known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

[0263] Stem cells are isolated for transduction and differentiationusing known methods. For example, stem cells are isolated from bonemarrow cells by panning the bone marrow cells with antibodies which bindunwanted cells, such as CD4+ and CD8+ (T cells), CD45+(panB cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

[0264] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can be also administered directlyto the organism for transduction of cells in vivo. Alternatively, nakedDNA can be administered.

[0265] Administration is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cells,as described below. The nucleic acids are administered in any suitablemanner, preferably with pharmaceutically acceptable carriers. Suitablemethods of administering such nucleic acids are available and well knownto those of skill in the art, and, although more than one route can beused to administer a particular composition, a particular route canoften provide a more immediate and more effective reaction than anotherroute (see Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989)). In particular, atleast six viral vector approaches are currently available for genetransfer in clinical trials, with retroviral vectors by far the mostfrequently used system. All of these viral vectors utilize approachesthat involve complementation of defective vectors by genes inserted intohelper cell lines to generate the transducing agent.

[0266] VIII. Pharmaceutical Compositions and Administration

[0267] p33ING2 nucleic acid, protein, and modulators of p33ING2 can beadministered directly to the patient for inhibition of cancer, tumor, orprecancer cells in vivo. Administration is by any of the routes normallyused for introducing a compound into ultimate contact with the tissue tobe treated. The compounds are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such compounds are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

[0268] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington 'sPharmaceutical Sciences, 17^(th) ed. 1985)). For example, if in vivodelivery of a biologically active p33ING2 protein is desired, themethods described in Schwarze et al. (see Science 285:1569-1572 (1999))can be used.

[0269] The compounds (nucleic acids, proteins, and modulators), alone orin combination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

[0270] Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

[0271] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular compound employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular compound or vector in a particularpatient

[0272] In determining the effective amount of the modulator to beadministered in the treatment or prophylaxis of cancer, the physicianevaluates circulating plasma levels of the modulator, modulatortoxicities, progression of the disease, and the production ofanti-modulator antibodies. In general, the dose equivalent of amodulator is from about 1 ng/kg to 10 mg/kg for a typical patient.Administration of compounds is well known to those of skill in the art(see, e.g., Bansinath et al., Neurochem Res. 18:1063-1066 (1993);Iwasaki et al., Jpn. J Cancer Res. 88:861-866 (1997); Tabrizi-Rad etal., Br. J. Pharmacol. 111:394-396 (1994)).

[0273] For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

[0274] IX. Diagnostics and Kits

[0275] The present invention also provides methods for detection ofp33ING2 (either wildtype or mutant). For example, kits are provided thatcontain p33ING2 specific reagents that specifically hybridize to p33ING2nucleic acid, such as specific probes and primers, and p33ING2 specificreagents that specifically bind to the protein of choice, e.g.,antibodies. The methods, kits, and the assays described herein can beused for identification of modulators of p33ING2, or for diagnosingpatients with mutations in p33ING2.

[0276] Nucleic acid assays for the presence of p33ING2 DNA and RNA in asample include numerous techniques are known to those skilled in theart. In particular, p33ING2 specific reagents (e.g., p33ING2-specificprimers or nucleic acid probes) can be used to distinguish betweensamples which contain p33ING2 nucleic acids and samples which containp33ING1 nucleic acids. Techniques such as Southern analysis, Northernanalysis, dot blots, RNase protection, high density oligonucleotidearrays, S1 analysis, amplification techniques such as PCR and LCR, andin situ hybridization can be used as assays. In in situ hybridization,for example, the target nucleic acid is liberated from its cellularsurroundings in such as to be available for hybridization within thecell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization: Singer et al., Biotechniques4:230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp.189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach(Hames et al., eds. 1987).

[0277] In addition, p33ING2 protein can be detected with the variousimmunoassay techniques described above, e.g., ELISA, western blotting,and the like. The test sample is typically compared to both a positivecontrol (e.g., a sample expressing recombinant p33ING2) and a negativecontrol. In particular, p33ING2 specific polyclonal and monoclonalantibodies or p33ING1 specific polyclonal and monoclonal antibodies canbe used as a diagnostic tool to distinguish between samples whichcontain p33ING2 antigens and samples which contain p33ING1 antigens.These polyclonal and monoclonal antibodies can also be used to determinethe amount of p33ING2 or p33ING1 in samples.

[0278] The present invention also provides for kits for screening formodulators of p33ING2. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: p33ING2, reaction tubes, andinstructions for testing p33ING2 activity. Preferably, the kit containsbiologically active p33ING2. Furthermore, the kit may include a label orwritten instructions for the use of one or more of these reagents andmaterials in any of the assays described herein. A wide variety of kitsand components can be prepared according to the present invention,depending upon the intended user of the kit and the particular needs ofthe user.

[0279] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0280] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

[0281] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

Example I Cloning and Expression of p33ING2

[0282] p33ING1 homologous sequences were found in a random cDNA sequencedatabase consisting of short partial sequences known as expressedsequence tags (ESTs) submitted in GenBank. Using primers designed basedon these EST sequences and using RT-PCR and 5′- and 3′-RACE methods,p33ING2 coding region (SEQ ID NO:2) from human placenta cDNA (CLONTECH)was isolated and subcloned into a plasmid.

[0283] To obtain a p33ING2 genomic sequence, a human PAC genomic librarywas screened with the p33ING2 cDNA sequence. Two clones were selected,one of which included the p33ING2 genomic sequence (SEQ ID NO:7;exon/intron). The genomic structure (exon/intron boundary sequence) wasdetermined by using human PAC genomic clones and “long distancesequence” method. The nucleic acid sequence of SEQ ID NO:10 isexon2/intron of p33ING2 genomic sequence.

[0284] Chromosomal localization of p33ING2 was determined using FISH(fluorescent in situ hybridization) analysis and human PAC genomic cloneincluding p33ING2 genome as a probe. It was determined that p33ING2 islocated at chromosome 4, at 4q35.

[0285] One human cancer cell line with a p33ING2 mutation, namely,HCT116, was discovered. As shown in SEQ ID NO:6, it has a missensemutation at amino acid position 153 (Arg to Ser).

Example II Cloning and Expression of p33ING1

[0286] The p33ING1 mRNA coding and amino acid sequences submitted inGenBank (Accession No. AF044076) had several mistakes. The correctsequence of p33ING1 mRNA coding region was determined by using humanplacental cDNA and RT-PCR method.

[0287] A human PAC genomic library was screened by p33ING1 cDNAsequence. Two clones were picked up which included p33ING1 genomicsequence. The genomic structure (exon/intron boundary sequence) wasdetermined by using the human PAC genomic clones and “long distancesequence” method. The sequence of mRNA coding sequence was alsoconfirmed by the genomic DNA sequence.

Example III Antibodies to p33ING2 and p33ING1

[0288] Antibodies to p33ING2 and p33ING1 were synthesized using twounique peptides (KMP-1 from p33ING2 (see, e.g., SEQ ID NO:5) and KMP-2from p33ING1 (see, e.g., SEQ ID NO:9)). These peptides were purified byHPLC; peptide KLH conjugations were made; and rabbits were immunized bythem. Antiserum was purified using peptide affinity column andspecificity of each polyclonal antibody was analyzed by ELISA.

[0289] As shown in FIG. 1, by ELISA (enzyme-linked immunosorbent assay)anti-p33ING2 polyclonal antibodies are reactive with recombinantGST-p33ING2 protein or its peptide fragment KMP-1 (SEQ ID NO:5), but arenot cross-reactive with recombinant GST-p33ING1 protein or its peptidefragment KMP-2 (SEQ ID NO:9). Anti-p33ING1 polyclonal antibodies arereactive with recombinant GST-p33ING1 protein or its peptide fragmentKMP-2, but are not cross-reactive with recombinant GST-p33ING2 proteinor its peptide fragment.

[0290] As shown in FIG. 2, by Western blot analysis, anti-p33ING2polyclonal antibodies are reactive with recombinant p33ING2 protein, butare not cross-reactive with recombinant p33ING1 protein. Anti-p33ING1polyclonal antibodies are reactive with recombinant p33ING1 protein, butare not cross-reactive with recombinant p33ING2 protein.

Example IV Inhibition of Cell Proliferation

[0291] The colony formation assay was used to determine if p33ING2inhibits cell growth of HCT116 cell line (human, hereditarynon-polyposis colon cancer cell line, wt p53).

[0292] Mammalian expression vectors (with CMV promoter, Neomycinresistant) containing p33ING2 in sense orientation (pcDNA3-ING2) and inantisense orientation (pcDNA3-AntiING2) were constructed. HCT116 celllines were transfected with the expression vectors. The transfectedcells were selected by Neomycin. The colony formation assay was used totest the effect of p33ING2 and anti-p33ING2 expression in HCT116 celllines. As shown in FIG. 3, HCT116 cells transfected with pcDNA3-ING2formed less colonies compared to HCT116 cells transfected withpcDNA3-AntiING2 or HCT116 cells transfected with pcDNA3 (without anyinserts). This result illustrates that p33ING2 inhibits cell growth.

Example V Soft Agar Assay for Identifying Compounds that Modulatep33ING2

[0293] Wildtype or mutant p33ING2 is expressed in host cells to screencompounds that modulate anchorage dependence of host cells expressingp33ING2. This is achieved by using the method disclosed in Garkavtsev etal. (1996), supra, herein incorporated by reference. Non-tumorigenicimmortalized mouse mammary epithelial cells (NMuMG) are transfected withretrovirus produced from a vector containing p33ING2 in sense orantisense orientation, or a vector lacking insert (control). The softagar culture is comprised of two layers: an underlay (DMEM, 10% FCS,0.6% agar) and an overlay (DMEM, 10% FCS, 0.3% agar), 5×10⁴ cells areplated in soft agar in 10 cm plates are left at 37° C. for 6-7 weeksbefore being counted. The cells are incubated with a test compound for asuitable amount of time, e.g., for 0.5 to 48 hours, before countingcells. The amount of cells in the test sample is then compared tocontrol cells untreated with the compound.

Example VI p33ING2 Protein Induction by DNA Damage

[0294] Calu6 cells were treated with topoisomerase II inhibitor,etoposide (SIGMA, E-1383, 10 μg/ml). Etoposide can induce DNA damage(e.g., double-strand DNA break). FIG. 4 shows the Western blot ofp33ING1, p33ING2 and beta-actin (as control). The protein analysisindicated that p33ING2 protein expression was induced by the treatmentof Calu6 cells with etoposide. However, p33ING1 protein expression wasnot induced by etopside.

Example VII p33ING1 or p33ING2 Can Induce G₁ Cell Cycle Arrest

[0295] RKO cells were transfected with pcDNA3.1 (control),pcDNA3.1-p33ING1, or pcDNA3.1-p33ING2. Cells were co-transfected withpEGFP—F Amp (a plasmid containing an enhanced green fluorescent proteinand an ampicillin transfection marker). The cells were gated by GFP. TheGFP-positive cells were considered to be pcDNA3.1, pcDNA3.1-p33ING1, orpcDNA3.1-p33ING2 positive. The propidium iodide signal was used as ameasure for DNA content to determine cell cycle profiles on a FACScanflow cytometer (Becton-Dickinson). See FIG. 5. The percentages of thecells in each cell cycle phage were calculated by the ModFit program(Becton-Dickinson), and the results are as follows:

[0296] pcDNA3.1 G₀/G₁ (43.1%), G2M (32.5%), S-phase (24.4%)

[0297] pcDNA3.1-p33ING1 G₀/G₁ (67.1%), G₂M (21.7%), S-phase (11.2%)

[0298] pcDNA3.1-p33ING2 G₀/G₁ (71.2%), G₂M (19.9%), S-phase (8.9%)

[0299] These results indicate that p33ING1 or p33ING2 can induce G₁ cellcycle arrest in cells.

Example VIII p33ING1 and p33ING2 can enhance p21/WAF1, BAX, and IGF BP3Promoter Activities in p53 Wild Type Cell Line RKO

[0300] The p53 transcriptional transactivities (p21/WAF1, BAX, or IGFBP3) were examined with the Dual-Luciferase Reporter Assay System(Promega, E1910). RKO cells were co-transfected with Renilla Luc vectorSV 40 (internal control) and p53 responsive reporter vectors,WWP-Luc-p21, PGL3-Luc-BAX, or pUHC13-3-Luc-IGF BP3 BOX B. The cells werealso transfected with pcDNA3.1, pcDNA3.1-p33ING1, or pcDNA3.1-p33ING2.The results of the promoter activity according the luciferase assay areas follows:

[0301] p21/WAF1 promoter activity (average+/−SD

[0302] pcDNA3.1 (100+/−9.3)

[0303] pcDNA3.1-p33ING1 (190.8+/−15.6)

[0304] pcDNA3.1-p33ING2 (199.8+/−29.2)

[0305] BAX promoter activity (average+/−SD

[0306] pcDNA3.1 (100+/−6.2)

[0307] pcDNA3.1-p33ING1 (237.6+/−15.4)

[0308] pcDNA3.1-p33ING2 (347.9+/−28.5)

[0309] IGF BP3 promoter activity (average+/−SD)

[0310] pcDNA3.1 (100+/−10.9)

[0311] pcDNA3.1-p33ING1 (181.8+/−20.6)

[0312] pcDNA3.1-p33ING2 (205.0+/−13.1)

[0313] The above results indicate that p33ING1 and p33ING2 enhancedp21/WAF1, BAX, and IGF BP3 promoter activities in p53 wild type cellline RKO.

Example IX p33ING1 and p33ING2 Induces Apoptosis

[0314] RKO cells were transfected with pcDNA3.1 (control),pcDNA3.1-p33ING1, or pcDNA3.1-p33ING2 expression vector. Cells wereco-transfected with pEGFP—F Amp (transfection marker). The cells werefixed 24 hours after transfection. The GFP-positive cells wereconsidered to be pcDNA3.1, pcDNA3.1-p33ING1, or pcDNA3.1-p33ING2positive. Apoptotic change was determined by DAPI staining and TUNELassay using fluorescent microscope. For TUNEL assay, the following kitand materials were used: Fluorescein FragEL DNA Fragmentation DetectionKit (Oncogene Research Products, Cat.#QIA39)+Tetramethyl-rhodamine-5-dUTP (Roche, Cat. # 1534 378).

[0315] The assay results are as follows.

[0316] % apoptotic cells/transfected cells (GFP-positive cells)

[0317] pcDNA3.1 (control): 15.3+/−1.3 (average+/−SD)

[0318] pcDNA3.1-p33ING1: 40.3+/−3.0

[0319] pcDNA3.1-p33ING2: 39.3+/−1.7

[0320] The above results indicate that expression or overexpression ofp33ING1 or p33ING2 induced apoptosis in RKO cells at a higher frequencycompared to the control.

1 10 1 280 PRT Artificial Sequence Description of Artificial Sequencep33ING2 polypeptide sequence 1 Met Leu Gly Gln Gln Gln Gln Gln Leu TyrSer Ser Ala Ala Leu Leu 1 5 10 15 Thr Gly Glu Arg Ser Arg Leu Leu ThrCys Tyr Val Gln Asp Tyr Leu 20 25 30 Glu Cys Val Glu Ser Leu Pro His AspMet Gln Arg Asn Val Ser Val 35 40 45 Leu Arg Glu Leu Asp Asn Lys Tyr GlnGlu Thr Leu Lys Glu Ile Asp 50 55 60 Asp Val Tyr Glu Lys Tyr Lys Lys GluAsp Asp Leu Asn Gln Lys Lys 65 70 75 80 Arg Leu Gln Gln Leu Leu Gln ArgAla Leu Ile Asn Ser Gln Glu Leu 85 90 95 Gly Asp Glu Lys Ile Gln Ile ValThr Gln Met Leu Glu Leu Val Glu 100 105 110 Asn Arg Ala Arg Gln Met GluLeu His Ser Gln Cys Phe Gln Asp Pro 115 120 125 Ala Glu Ser Glu Arg AlaSer Asp Lys Ala Lys Met Asp Ser Ser Gln 130 135 140 Pro Glu Arg Ser SerArg Arg Pro Arg Arg Gln Arg Thr Ser Glu Ser 145 150 155 160 Arg Asp LeuCys His Met Ala Asn Gly Ile Glu Asp Cys Asp Asp Gln 165 170 175 Pro ProLys Glu Lys Lys Ser Lys Ser Ala Lys Lys Lys Lys Arg Ser 180 185 190 LysAla Lys Gln Glu Arg Glu Ala Ser Pro Val Glu Phe Ala Ile Asp 195 200 205Pro Asn Glu Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu 210 215220 Met Ile Gly Cys Asp Asn Glu Gln Cys Pro Ile Glu Trp Phe His Phe 225230 235 240 Ser Cys Val Ser Leu Thr Tyr Lys Pro Lys Gly Lys Trp Tyr CysPro 245 250 255 Lys Cys Arg Gly Asp Asn Glu Lys Thr Met Asp Lys Ser ThrGlu Lys 260 265 270 Thr Lys Lys Asp Arg Arg Ser Arg 275 280 2 1080 DNAArtificial Sequence Description of Artificial Sequence p33ING2 nucleicacid sequence (GenBank Accession No. AF05053537) 2 gcggccgcgg ccggtgcatgtgcggctgct ggatgcggag gcggcggcga cggcgcggat 60 cggcaggatg ttagggcagcagcagcagca actgtactcg tcggctgcgc tcctgaccgg 120 ggagcggagc cggctgctcacctgctacgt gcaggactac cttgagtgcg tggagtcgct 180 gccccacgac atgcagaggaacgtgtctgt gctgcgagag ctggacaaca aatatcaaga 240 aacgttaaag gaaattgatgatgtctacga aaaatataag aaagaagatg atttaaacca 300 gaagaaacgt ctacagcagcttctccagag agcactaatt aatagtcaag aattgggaga 360 tgaaaaaata cagattgttacacaaatgct cgaattggtg gaaaatcggg caagacaaat 420 ggagttacac tcacagtgtttccaagatcc tgctgaaagt gaacgagcct cagataaagc 480 aaagatggat tccagccaaccagaaagatc ttcaagaaga ccccgcaggc agcggaccag 540 tgaaagccgt gatttatgtcacatggcaaa tgggattgaa gactgtgatg atcagccacc 600 taaagaaaag aaatccaagtcagcaaagaa aaagaaacgc tccaaggcca agcaggaaag 660 ggaagcttca cctgttgagtttgcaataga tcctaatgaa cctacatact gcttatgcaa 720 ccaagtgtct tatggggagatgataggatg tgacaatgaa cagtgtccaa ttgaatggtt 780 tcacttttca tgtgtttcacttacctataa accaaagggg aaatggtatt gcccaaagtg 840 caggggagat aatgagaaaacaatggacaa aagtactgaa aagacaaaaa aggatagaag 900 atcgaggtag taaaggccatccacatttta aagggttatt tgtcttttat ataattcgtt 960 tgctttcaga aaatgttttagggtaaatgc ataagactat gcaataattt ttaatcatta 1020 gtattaatgg tgtattaaaagttgttgtac tttgaaaaaa aaaaaaaaaa aaaaaaaaaa 1080 3 7 PRT ArtificialSequence Description of Artificial Sequence Degenerate primer used toisolate p33ING2 nucleic acids 3 Met Leu Gly Gln Gln Gln Gln 1 5 4 7 PRTArtificial Sequence Description of Artificial Sequence Degenerate primerused to isolate p33ING2 nucleic acid 4 Lys Lys Asp Arg Arg Ser Arg 1 5 520 PRT Artificial Sequence Description of Artificial Sequence peptide7-26 of p33ING2 (KMP1) 5 Gln Gln Leu Tyr Ser Ser Ala Ala Leu Leu Thr GlyGlu Arg Ser Arg 1 5 10 15 Leu Leu Thr Cys 20 6 280 PRT ArtificialSequence Description of Artificial Sequence missense p33ING2 sequence -Arg 153 to Ser 6 Met Leu Gly Gln Gln Gln Gln Gln Leu Tyr Ser Ser Ala AlaLeu Leu 1 5 10 15 Thr Gly Glu Arg Ser Arg Leu Leu Thr Cys Tyr Val GlnAsp Tyr Leu 20 25 30 Glu Cys Val Glu Ser Leu Pro His Asp Met Gln Arg AsnVal Ser Val 35 40 45 Leu Arg Glu Leu Asp Asn Lys Tyr Gln Glu Thr Leu LysGlu Ile Asp 50 55 60 Asp Val Tyr Glu Lys Tyr Lys Lys Glu Asp Asp Leu AsnGln Lys Lys 65 70 75 80 Arg Leu Gln Gln Leu Leu Gln Arg Ala Leu Ile AsnSer Gln Glu Leu 85 90 95 Gly Asp Glu Lys Ile Gln Ile Val Thr Gln Met LeuGlu Leu Val Glu 100 105 110 Asn Arg Ala Arg Gln Met Glu Leu His Ser GlnCys Phe Gln Asp Pro 115 120 125 Ala Glu Ser Glu Arg Ala Ser Asp Lys AlaLys Met Asp Ser Ser Gln 130 135 140 Pro Glu Arg Ser Ser Arg Arg Pro SerArg Gln Arg Thr Ser Glu Ser 145 150 155 160 Arg Asp Leu Cys His Met AlaAsn Gly Ile Glu Asp Cys Asp Asp Gln 165 170 175 Pro Pro Lys Glu Lys LysSer Lys Ser Ala Lys Lys Lys Lys Arg Ser 180 185 190 Lys Ala Lys Gln GluArg Glu Ala Ser Pro Val Glu Phe Ala Ile Asp 195 200 205 Pro Asn Glu ProThr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu 210 215 220 Met Ile GlyCys Asp Asn Glu Gln Cys Pro Ile Glu Trp Phe His Phe 225 230 235 240 SerCys Val Ser Leu Thr Tyr Lys Pro Lys Gly Lys Trp Tyr Cys Pro 245 250 255Lys Cys Arg Gly Asp Asn Glu Lys Thr Met Asp Lys Ser Thr Glu Lys 260 265270 Thr Lys Lys Asp Arg Arg Ser Arg 275 280 7 423 DNA Homo sapiens exon(1)..(239) p 33ING2 genomic DNA sequence (exon 1/intron) GenBankAccession No. HSING2S1 7 gcggccgcgg ccggtgcatg tgcggctgct ggatgcggaggcggcggcga cggcgcggat 60 cggcaggatg ttagggcagc agcagcagca actgtactcgtcggctgcgc tcctgaccgg 120 ggagcggagc cggctgctca cctgctacgt gcaggactaccttgagtgcg tggagtcgct 180 gccccacgac atgcagagga acgtgtctgt gctgcgagagctggacaaca aatatcaagg 240 taggggccgc ggggctgccg gcctcgggag ccggtggcggggagcctgtc cgggggagtg 300 ccaccttccc tttctcccgt gacagtctcc ccgagcgcaccgagggtctg ccgagcggga 360 ctgggaggac tggagaccgg gttggcggcc ctccgtggccccgcggtggg cgagtgaagg 420 aga 423 8 279 PRT Artificial SequenceDescription of Artificial Sequence p33ING1 8 Met Leu Ser Pro Ala Asn GlyGlu Gln Leu His Leu Val Asn Tyr Val 1 5 10 15 Glu Asp Tyr Leu Asp SerIle Glu Ser Leu Pro Phe Asp Leu Gln Arg 20 25 30 Asn Val Ser Leu Met ArgGlu Ile Asp Ala Lys Tyr Gln Glu Ile Leu 35 40 45 Lys Glu Leu Asp Glu CysTyr Glu Arg Phe Ser Arg Glu Thr Asp Gly 50 55 60 Ala Gln Lys Arg Arg MetLeu His Cys Val Gln Arg Ala Leu Ile Arg 65 70 75 80 Ser Gln Glu Leu GlyAsp Glu Lys Ile Gln Ile Val Ser Gln Met Val 85 90 95 Glu Leu Val Glu AsnArg Thr Arg Gln Val Asp Ser His Val Glu Leu 100 105 110 Phe Glu Ala GlnGln Glu Leu Gly Asp Thr Ala Gly Asn Ser Gly Lys 115 120 125 Ala Gly AlaAsp Arg Pro Lys Gly Glu Ala Ala Ala Gln Ala Asp Lys 130 135 140 Pro AsnSer Lys Arg Ser Arg Arg Gln Arg Asn Asn Glu Asn Arg Glu 145 150 155 160Asn Ala Ser Ser Asn His Asp His Asp Asp Gly Ala Ser Gly Thr Pro 165 170175 Lys Glu Lys Lys Ala Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Ala 180185 190 Lys Ala Glu Arg Glu Ala Ser Pro Ala Asp Leu Pro Ile Asp Pro Asn195 200 205 Glu Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu MetIle 210 215 220 Gly Cys Asp Asn Asp Glu Cys Pro Ile Glu Trp Phe His PheSer Cys 225 230 235 240 Val Gly Leu Asn His Lys Pro Lys Gly Lys Trp TyrCys Pro Lys Cys 245 250 255 Arg Gly Glu Asn Glu Lys Thr Met Asp Lys AlaLeu Glu Lys Ser Lys 260 265 270 Lys Glu Arg Ala Tyr Asn Arg 275 9 279PRT Artificial Sequence Description of Artificial Sequence peptide 1-17and C of p33ING1 (KMP2) 9 Met Leu Ser Pro Ala Asn Gly Glu Gln Leu HisLeu Val Asn Tyr Val 1 5 10 15 Glu Asp Tyr Leu Asp Ser Ile Glu Ser LeuPro Phe Asp Leu Gln Arg 20 25 30 Asn Val Ser Leu Met Arg Glu Ile Asp AlaLys Tyr Gln Glu Ile Leu 35 40 45 Lys Glu Leu Asp Glu Cys Tyr Glu Arg PheSer Arg Glu Thr Asp Gly 50 55 60 Ala Gln Lys Arg Arg Met Leu His Cys ValGln Arg Ala Leu Ile Arg 65 70 75 80 Ser Gln Glu Leu Gly Asp Glu Lys IleGln Ile Val Ser Gln Met Val 85 90 95 Glu Leu Val Glu Asn Arg Thr Arg GlnVal Asp Ser His Val Glu Leu 100 105 110 Phe Glu Ala Gln Gln Glu Leu GlyAsp Thr Ala Gly Asn Ser Gly Lys 115 120 125 Ala Gly Ala Asp Arg Pro LysGly Glu Ala Ala Ala Gln Ala Asp Lys 130 135 140 Pro Asn Ser Lys Arg SerArg Arg Gln Arg Asn Asn Glu Asn Arg Glu 145 150 155 160 Asn Ala Ser SerAsn His Asp His Asp Asp Gly Ala Ser Gly Thr Pro 165 170 175 Lys Glu LysLys Ala Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Ala 180 185 190 Lys AlaGlu Arg Glu Ala Ser Pro Ala Asp Leu Pro Ile Asp Pro Asn 195 200 205 GluPro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Ile 210 215 220Gly Cys Asp Asn Asp Glu Cys Pro Ile Glu Trp Phe His Phe Ser Cys 225 230235 240 Val Gly Leu Asn His Lys Pro Lys Gly Lys Trp Tyr Cys Pro Lys Cys245 250 255 Arg Gly Glu Asn Glu Lys Thr Met Asp Lys Ala Leu Glu Lys SerLys 260 265 270 Lys Glu Arg Ala Tyr Asn Arg 275 10 974 DNA Homo sapiensintron (<1)..(123) p33ING2 genomic DNA sequence (Exon 2/intron) GenBankAccession No. HSING2S2 10 ccaaagagga gtatggtttc atggtttgag ttctaatttcaattctgtaa aaaataacta 60 ccttggaaat gttgtgtctg ctaacacatg ataacgttctcatttttctt ttcctttttt 120 tagaaacgtt aaaggaaatt gatgatgtct acgaaaaatataagaaagaa gatgatttaa 180 accagaagaa acgtctacag cagcttctcc agagagcactaattaatagt caagaattgg 240 gagatgaaaa aatacagatt gttacacaaa tgctcgaattggtggaaaat cgggcaagac 300 aaatggagtt acactcacag tgtttccaag atcctgctgaaagtgaacga gcctcagata 360 aagcaaagat ggattccagc caaccagaaa gatcttcaagaagaccccgc aggcagcgga 420 ccagtgaaag ccgtgattta tgtcacatgg caaatgggattgaagactgt gatgatcagc 480 cacctaaaga aaagaaatcc aagtcagcaa agaaaaagaaacgctccaag gccaagcagg 540 aaagggaagc ttcacctgtt gagtttgcaa tagatcctaatgaacctaca tactgcttat 600 gcaaccaagt gtcttatggg gagatgatag gatgtgacaatgaacagtgt ccaattgaat 660 ggtttcactt ttcatgtgtt tcacttacct ataaaccaaaggggaaatgg tattgcccaa 720 agtgcagggg agataatgag aaaacaatgg acaaaagtactgaaaagaca aaaaaggata 780 gaagatcgag gtagtaaagg ccatccacat tttaaagggttatttgtctt ttatataatt 840 cgtttgcttt cagaaaatgt tttagggtaa atgcataagactatgcaata atttttaatc 900 attagtatta atggtgtatt aaaagttgtt gtactttgtctgtgacctta attttctgca 960 ctgagttacc aaat 974

1-14. (Canceled)
 15. An antibody that binds only to a p33ING2polypeptide comprising an amino acid sequence of SEQ ID NO:1, but doesnot bind to a p33ING1 polypeptide comprising an amino acid sequence ofSEQ ID NO:8.
 16. The antibody of claim 15, wherein the antibody ispolyclonal. 17-18. (Canceled)
 19. The antibody of claim 15, wherein theantibody binds only to a p33ING2 polypeptide comprising the amino acidsequence of SEQ ID NO:5, but does not bind to a p33ING1 polypeptidecomprising an amino acid sequence of SEQ ID NO:8. 20-57. (Canceled) 58.The antibody of claim 15, wherein the antibody is a monoclonal antibody.59. The antibody of claim 15, wherein the antibody is a humanizedantibody.
 60. The antibody of claim 15, wherein the antibody is achimeric antibody.
 61. A method of making the antibody of claim 15, themethod comprising the steps of: a) injecting an animal with a purifiedp33ING2 polypeptide comprising an amino acid sequence of SEQ ID NO:1 oran antigenic fragment thereof, wherein the animal produces the p33ING2antibody from an immune cell, and b) collecting a biological sample thatcomprises the p33ING2 antibody from the animal.
 62. The method of claim61, further comprising the step of isolating the p33ING2 antibody fromthe biological sample.
 63. The method of claim 61, wherein the antibodyis a polyclonal antibody and the biological sample is serum.
 64. Themethod of claim 61, wherein a purified antigenic fragment of the p33ING2 polypeptide is injected into the animal.
 65. The method of claim 64,wherein the purified antigenic fragment of the p33ING 2 polypeptide isSEQ ID NO:5.
 66. The method of claim 61, wherein the animal is a mouse,the biological sample comprises spleen cells, and the antibody is amonoclonal antibody, the method further comprising the step of fusingthe spleen cell with an immortalized cell to produce the monoclonalantibody.